Selective recovery

ABSTRACT

Provided herein are methods of selective screening. In addition, various targeting proteins and sequences, as well as methods of their use, are also provided.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/485,024, filed Sep. 12, 2014, which claims priority to U.S.Provisional Application No. 61/877,506, filed Sep. 13, 2013, U.S.Provisional Application No. 61/983,624, filed Apr. 24, 2014, U.S.Provisional Application No. 62/020,658, filed Jul. 3, 2014, and U.S.Provisional Application No. 62/034,060, filed Aug. 6, 2014, each ofwhich is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under Grant No.MH100556, Grant No. MH086383, Grant No. AG047664 and Grant No. 0D017782awarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING IN ELECTRONIC FORMAT

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSEQUENCELISTING_CALTE103C1.TXT, created on Oct. 21, 2014, last modifiedon Feb. 1, 2017, which is 98,622 bytes in size. The information in theelectronic format of the Sequence Listing is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of selective recovery andtargeting proteins and/or methods.

BACKGROUND

Recombinant adeno-associated viruses (rAAV) are vectors for in vivo genetransfer applications. Several rAAV-based gene therapies are proving tobe efficacious, most notably for the treatment of Leber's congenitalamaurosis, hemophilia associated with factor IX deficiency andlipoprotein lipase deficiency (Simonelli et al 2010; Nathwani et al2011; Gaudet et al. 2010). Recently, the first rAAV-based gene therapy,Glybera, was approved by the European Medicines Agency for the treatmentof lipoprotein lipase deficiency. rAAVs have also shown success inpreclinical models of a large variety of diseases, including Rettsyndrome, congenital ALS, Parkinson's, Huntington's disease, SpinalMuscular Atrophy, among others and for the prophylactic delivery ofbroad neutralizing antibodies against infectious diseases such as HIVand influenza (Garg et al 2013; Valori et al. 2010; Foust et al. 2010;Foust et al 2013; Southwell et al 2009; Balazs et al 2011; and Balazs etal 2013). In addition, rAAVs are also popular vectors for in vivodelivery of transgenes for non-therapeutic scientific studies, such asoptogenics.

SUMMARY OF THE INVENTION

In some embodiments, an AAV vector is provided that comprises an aminoacid sequence that comprises at least 4 contiguous amino acids from thesequence TLAVPFK (SEQ ID NO: 1) or KFPVALT (SEQ ID NO: 3).

In some embodiments, a central nervous system targeting peptide isprovided that comprises an amino acid sequence of SEQ ID NO: 1 (or anyof the amino acid sequences in FIG. 31).

In some embodiments, a nucleic acid sequence encoding any fourcontiguous amino acids in TLAVPFK (SEQ ID NO: 1) or in KFPVALT (SEQ IDNO: 3) is provided (or for any of the sequences from FIG. 31).

In some embodiments, a method of delivering a nucleic acid sequence to anervous system is provided. The method can comprise providing a proteincomprising TLAVPFK (SEQ ID NO: 1) (or any of the other targetingsequences provided herein, for example, in FIG. 31), wherein the proteinis part of a capsid of an AAV, and wherein the AAV comprises a nucleicacid sequence to be delivered to a nervous system; and administering theAAV to the subject.

In some embodiments, an rAAV genome is provided that comprises at leastone inverted terminal repeat configured to allow packaging into a vectorand a cap gene.

In some embodiments a plasmid system is provided that comprises a firstplasmid comprising a modified AAV2/9 rep-cap helper plasmid configuredsuch that it eliminates at least one of VP1, VP2, or VP3 expression anda second plasmid comprising a rAAV-cap-in-cis plasmid.

In some embodiments, a method of developing a capsid with a desiredcharacteristic is provided. The method can comprise providing apopulation of rAAV genomes (of any provided herein), selecting thepopulation by a specific set of criteria, and selecting the rAAV genomethat meets the screening criteria.

In some embodiments, a capsid protein is provided that comprises anamino acid sequence that comprises at least 4 contiguous amino acidsfrom the sequence TLAVPFK (SEQ ID NO: 1) or KFPVALT (SEQ ID NO: 3) (orfrom any of the sequences in FIG. 31).

In some embodiments, a library of nucleic acid sequences is provided.The library can comprise a selectable element and one or morerecombinase recognition sequences.

In some embodiments, a method of developing a capsid with a desiredcharacteristic is provided. The method comprises providing a library ofplasmids that comprise a capsid gene, and at least one recombinaserecognition sequence, configured such that it allows arecombinase-dependent change in a sequence of a plasmid of the librarythat comprises the capsid gene that is a detectable change. The methodcan further comprise selecting the population by a specific set ofcriteria and selecting the rAAV genome that meets the screeningcriteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts various embodiments for aspects of various targetingproteins (in this case, examples of AAV capsid proteins), includingG2B-13, G2B-26, TH1.1-32 and TH1.1-35.

FIG. 1B depicts some embodiments of selective recovery of capsidproteins.

FIG. 2A depicts some embodiments of AAV genome manipulation.

FIG. 2B depicts some embodiments of capsid gene manipulation.

FIG. 2C depicts a flow chart for some embodiments of selective recoveryembodiments.

FIG. 2D depicts a schematic of AAV genes and their known products. TheAAV rep gene makes four protein products shown in black. The cap genemakes three structural capsid proteins (VP1-3) from one reading frame bya combination of alternative splicing and alternative initiator codons.In addition, the capsid gene also encodes an additional protein,assembly-activating protein (AAP), which is expressed from analternative reading frame.

FIG. 2E is a schematic for the design of constructs used for someembodiments of the rAAV-based capsid library selection method. A capsidgene is inserted within a recombinant AAV genome flanked by ITRs. Theexpression and splicing of the AAV capsid gene products is controlled bythe AAV5 p41 promoter upstream of the AAV2 rep sequences that containthe splice donor and intron sequences for the capsid gene products. Byeliminating most of the rep gene, space (represented by the dottedlines) is available within the rAAV cap-in-cis genome for the insertionof additional elements.

FIG. 2F is a schematic showing the AAV components of the rep-AAP helperplasmid. Five stop codons were inserted within the capsid VP readingframe to ensure that VP1, VP2 and VP3 expression is eliminated from therep-AAP helper.

FIGS. 3A and 3B. A strategy for recombinase-dependent recovery ofsequences from transduced target cells. (FIG. 3A) The schematic showsthe cap-in-cis rAAV genome. A ubiquitin C promoter fragment can be usedto drive expression of an mCherry reporter followed by a synthetic polyAsequence. An AAV capsid gene, controlled by rep regulatory sequences, isfollowed by a lox71- and lox66-flanked SV40 late polyA signal. The lox66site is inverted relative to lox71. In this configuration, Cre mediatesthe inversion (FIG. 3B) of the sequence flanked by the mutant lox sites.After the inversion, incompatible, double mutant lox72 and a loxP siteare generated, reducing the efficiency of inversion back to the originalstate. Using PCR primers represented by the arrows in the schematic, capsequences can be recovered selectively from genomes that have undergonea Cre-dependent inversion.

FIGS. 4A-4D. Alternative lox strategies for recombinase dependentrecovery. (FIG. 4A) Single inverted loxP or lox71 and lox66 sites can bereplaced by double loxP (white triangle) and lox2272 (black triangle)for irreversible recombination. (FIG. 4B) loxP sites or similar sites(lox2272 or other variants) inserted in the same orientation to mediatea deletion also allow selective recombination-dependent amplification.Recombination specific recovery can be achieved by performing thePCR-based recovery with three primers: one 5′ of the randomized sequence(black arrow), one reverse primer downstream of the 3′ loxP sequence(dark gray arrow) and a second forward primer that binds specificallywithin the deleted sequence (light gray arrow). This primer out competesthe 5′ (black) forward primer during amplification, reducingamplification of cap sequences from non-recombined sequences. Recoveryof non-recombined sequences can also be reduced by digestion with anenzyme that recognizes a site within the sequence deleted by therecombinase. Alternatively, Cre-dependent and -independent products canbe separated by size by gel electrophoresis. (FIGS. 4C & 4D) InvertedloxP, lox71 and lox66 (shown), or DIO, FLEX sites can be placed inalternative configurations. (FIG. 4C) shows lox sites in an invertedorientation surrounding the rep and cap sequences, which can be invertedin the presence of Cre. (FIG. 4D) Schematic shows the option of flankingthe reporter with lox sites. In this embodiment, the reporter isinverted and can be expressed after recombination.

FIGS. 5A-5C. A split Rep/AAP helper and rAAV-Cap-lox vector produceshigh titer virus. (FIG. 5A) DNase-resistant AAV genome copies (GCs)produced with the split AAV2/9 rep-AAP and AAV9 cap-in-cis genome(left), the AAV2/9 rep-AAP and a mCherry expressing rAAV genome (nocap-middle) or a control AAV2/9 rep/cap helper with the sameAAV2:mCherry genome (right). (FIG. 5B) DNase-resistant viral GCsobtained from larger scale (7-10 150 mm plate) preps of libraries withrandomized 7-mer sequences replacing AAV9 capsid amino acids 452-8(left) or inserted after amino acid 588 (right) (n=4/per library±SEM).(FIG. 5C) The PCR fragments containing the capsid sequence variation(black, 452-8 or light gray, 588) libraries are generated and clonedinto a rAAV9R-delta-X/Acap-in-cis vector that has been modified toinsert unique restriction sites XbaI (X) and AgeI (A) flanking theregion to be modified.

FIGS. 6A-6C. Cre-dependent sequence recovery after selection in Cretransgenics or Cre+ cells. (FIG. 6A) The schematic shows an overview ofthe selection process. In example 3, GFAP-Cre+ mice were injected withAAV virus containing AAV9-cap-in-cis, or the cap libraries with random7mers at amino acids 452-8 or 588, and PCR products were recovered usingprimers that selectively amplify sequences from cap-in-cis genomes thathave undergone Cre-mediated inversion of the sequence 3′ to the capgene. (FIG. 6B) The image shows an ethidium bromide-stained agarose gelof the PCR products recovered after the second PCR step using primers1331 and 1312. (FIG. 6C) Recovered PCR products are then cloned into therAAV9R-delta-X/A-cap-in-cis vector as a first step to generate the nextround of capsid virus libraries.

FIG. 7. The novel AAV variants generate virus with efficiencies similarto AAV9. The graph shows the DNase-resistant viral GCs generated per 150mm dish of near confluent 293 producer cells for AAV9 and the four novelAAV serotypes.

FIGS. 8A-8C. G2B13 and G2B26 variants mediate enhanced transduction ofthe brain and spinal cord after IV administration as compared to AAV9.An AAV-CAG-eGFP-2A-ffLUC-WPRE-SV40 pA vector was packaged into AAV9(left) or the novel variants G2B13 (middle) or G2B26 (right). 1e12 GC ofeach virus was injected IV into individual 5-week old female wt C57B1/6mice and the brains of the mice were assessed for GFP expression 6 dayslater. (FIG. 8A) Panels show native eGFP fluorescence in whole brain.(FIG. 8B) Immunostaining for eGFP expression in the sectioned brains ofmice injected with the indicated virus show efficient transduction ofmultiple cell types including neurons (n) and astrocytes (a). (FIG. 8C)Panels show native eGFP fluorescence in the livers of mice injected withthe indicated virus.

FIGS. 9A-9I. G2B13 and G2B26 variants mediate enhanced transduction ofCNS neurons and glia after IV administration as compared to AAV9. ArAAV-CAG-eGFP-2A-ffLUC-WPRE-SV40-pA vector was packaged into G2B13 (FIG.9A-9C) or G2B26 (FIGS. 9D-9I) and 1e12 GC of the indicated virus wasinjected IV into individual 5-week old female wt C57B1/6 mice. (FIG.9A-9B, FIG. 9D-9I) Panels show immunostaining for eGFP in the sectionedbrains of mice 6 days after they were injected IV withG2B13:CAG-GFP2A-Luc (FIG. 9A-9B) or G2B26:CAG-GFP2A-Luc (FIG. 9D, FIG.9E, FIG. 9G-9I). Both vectors show transduction of several cell typesincluding neurons (n) and astrocytes (a). (FIG. 9A-9B, FIG. 9D-9E, FIG.9G-911) Panels show eGFP immunostaining (upper panels) and NeuNimmunostaining (lower panels) from paired image fields. (FIG. 9C andFIG. 9F) Panels show native eGFP fluorescence in the spinal cords ofmice injected with G2B13 (FIG. 9C) or G2B26 (FIG. 9F). (FIG. 9I) Panelshows eGFP immunostaining in (upper panels) co-localized (arrows) withthe glial marker Sox2 (lower panels) from the same image field.

FIG. 10. Strategy for generating further diversity by combiningsequences recovered at multiple sites. Following one or more rounds ofselection for novel cap variants at two different sites, the pools ofselected variants can be mixed to generate libraries that combine therandomized sequences at two or more sites by overlapping PCR. Using thesame strategy, individual clones with novel sequences at 2 more sitescan also be combined to generate clones with multiple modifications.

FIGS. 11A-11E. Cre-dependent sequence recovery after in vivo selection.(FIG. 11A) The Brain Explorer 2 (Allen Brain Atlas) schematic shows anoverview of the selection process. Capsid virus libraries were injectedbilaterally into the straita of TH-Cre+ mice (asterisks show approximateinjection sites), and capsid sequences were recovered from thesubstantia nigra (highlighted with a white square). (FIG. 11B) The imageshows native mCherry fluorescence from 1 mm slices through the forebraincontaining the striatal injection sites. (FIG. 11C) mCherry+ fibers fromstriatal neurons can be seen in slices from the SNr. The SNr and SNclocated dorsal to the SNr were collected for capsid sequence recovery.(FIG. 11D) Panels show ethidium bromide stained PCR products recoveredfrom TH-Cre+ cells of mice injected with AAV virus containing thelibraries with random 7mers at amino acids 452-8 or 588 (round 1). (FIG.11E) Cap-in-cis genomes were recovered by a Cre-independent PCR strategyfrom Cre+ and Cre−mice demonstrating the presence of virus in allsamples.

FIGS. 12A-12H. TH1.1-32 and -35 variants exhibit rapid and efficientretrograde transduction of TH+ SNc neurons as well as neurons inadditional regions known to project to the striatum.AAV-TH1.1-32:CAG-GFP or AAVTH1.1-35:CAG-GFP were injected into thestriatum of adult mice and mice were killed 7 days later for GFPexpression analysis. Panels show immunostaining for eGFP (FIG. 12A-B,FIG. 12D and FIG. 12F-H) or TH (FIG. 12C and FIG. 12E). (FIG. 12A) GFPexpression within the striatum surrounding the injection site of theTH1.1-35 variant. (FIG. 12B-E) Panels show GFP immunostaining (FIG. 12Band FIG. 12D) and TH immunostaining (FIG. 12C and FIG. 12E) within thesame image field within the SN. Co-localization of GFP and TH+immunostaining within the same cell is noted with arrows. GFP expressionis evident in the SNr and a subpopulation of TH+ neurons in the SNc of amouse injected with TH1.1-35 (FIG. 12B-C) or TH1.1-32 (FIG. 12D-E).(FIG. 12F) GFP immunostaining in the frontal cortex of a mouse injectedwith the TH1.1-35 variant. GFP immunostaining in the thalamus (FIG. 12G)and amygdala (FIG. 12H) of a mouse injected with the TH1.1-32 variant.

FIG. 13 depicts a sequence alignment of AAVs and related parvovirusesshowing diversity at 7mer insertion/replacement sites. The figure issplit into three parts, with the first sheet representing the left-handside of the figure, the second sheet representing the middle part of thefigure, and the third sheet of FIG. 13 representing the right-hand sideof the sheet. That is, the names for each of the rows in the first sheetare intended to carry across to the other two sheets (in each row).

FIG. 14 depicts a structural model of some embodiments of a capsidprotein showing the loop regions where targeting sequences can be addedor substituted in.

FIG. 15 depicts some embodiments of the rAAV9R-delta-X/A-cap-in-cisvector.

FIG. 16 depicts some embodiments of an AAV rep/cap helper plasmid thatwas modified by inserting a total of 5 stop codons within the cap genewithin the VP1, 2 and 3 reading frame (1 stop codon disrupts VP3, 3disrupt VP2 and all 5 disrupt VP1—FIG. 2F, SEQ ID NO: 5).

FIG. 17 depicts some embodiments of a template DNA (pCRII-9R-X/A EKplasmid, SEQ ID NO: 6.)

FIG. 18 depicts AAV9R-delta-X/A-cap-in-cis, SEQ ID NO: 7, in which thecoding region between the XbaI and AgeI sites was eliminated to prevent“wt” AAV9R X/A capsid protein production from any undigested vectorduring library virus production.

FIG. 19 depicts some embodiments of a sequence of an AAV-PHP.B(AAV-G2B26) capsid VP1 protein.

FIG. 20 depicts some embodiments of a nucleic acid sequence for anAAV-PHP.B (AAV-G2B26) capsid gene coding sequence.

FIG. 21 depicts some embodiments in which a nucleic acid sequence for atargeting protein is cloned into an AAV Rep-Cap helper plasmid.

FIGS. 22A-22F. Recombinase-dependent recovery of AAV capsid sequencesfrom transduced target cells. FIG. 22A is a schematic showing therAAV-cap-in-cis rAAV genome used for capsid library generation. Cremediates the inversion of the sequence flanked by the mutant lox sitesand PCR primers, represented by half arrows in the schematic, are usedto selectively amplify the recombined sequences. (FIG. 22B) Theschematic shows the AAV components of the Rep-AAP helper plasmid. Stopcodons inserted in the VP reading frame eliminate VP1, VP2 and VP3.(FIG. 22C) DNase-resistant AAV genome copies (GCs) produced with thesplit AAV2/9 rep-AAP and AAV9 cap-in-cis genome (left) as compared to acontrol AAV2/9 rep/cap helper with a control AAV-UBC-mCherry genome(middle) or the AAV2/9 rep-AAP and control AAV-UBC-mCherry genome (nocap-right). (FIG. 22D) Representative PCR products showing Cre dependent(top) and Cre independent (bottom) amplification of recovered capsidlibrary sequences from TH-Cre positive or Cre negative mice. (FIG. 22E)The capsid sequence variation libraries at AA452-8 or after AA588 ofAAV9 (vertical gradient) are generated by PCR and cloned into arAAV-Δcap-in-cis vector that has been modified to insert uniquerestriction sites XbaI (x) and AgeI (a) flanking the variable region(s).(FIG. 22F) The schematic shows an overview of some embodiments of theselection process.

FIGS. 23A-23I. AAV-PHP.R2 mediates rapid and efficient retrogradetransduction. (FIG. 23A) Capsid libraries were injected into thestriatum (left) and tissue from the substantia nigra was collected forcapsid sequence recovery 10 days later. (FIG. 23A) Images show nativemCherry fluorescence expressed from the cap-in-cis library genomessurrounding the injection sites (left) and mCherry positive axons fromstriatal neurons in the SNr (right). (FIG. 23B-FIG. 23H) The recoveredAAV variant PHP.R2 was used to package ssAAV-CAG-GFP and 7×10⁹ VG wasinjected into the striatum. Seven days later, the mice were assessed forGFP expression by immunostaining. The images show GFP positive cells atthe striatal injection site (FIG. 23B) or at the indicated brain regionsthat contain GFP+ cell bodies (FIG. 23C, FIG. 23E-I). (FIG. 23D) showsimmunostaining for TH in the SNc. (FIG. 23E) shows GFP immunostainingfrom the same field shown in FIG. 23D. Co-localization of GFP and THimmunostaining within the same cell is noted with arrows. Scale bars are100 um in C, E-H and 20 um in 23D.

FIGS. 24A-24I. AAV-PHP.B mediates robust transduction of the entire CNSafter IV administration. Representative images from mice transduced with1×10¹² VG of ssAAV-CAG-GFP-2A-Luc packaged in AAV9 or AAV-PHP.B. GFPexpression was assessed 3 weeks later by immunostaining (FIG. 24A andFIG. 24C) or native GFP fluorescence (FIG. 24B, FIG. 24D-24G). (FIG.24A) Images show GFP immunostaining in sagittal brain sections from micegiven AAV9 (left), an equivalent dose of AAV-PHP.B (middle) or 1×10¹¹ VGof AAV-PHP.B (right). (FIG. 24B) Representative cortical (left) orstriatal (right) 50 um maximum confocal projection images of native eGFPfluorescence from the brains of mice treated as in 24A. (FIG. 24C)Nearly all Calbindin⁺ Purkinje cells (bottom) are GFP⁺ (top) 3 weeksafter IV injection of 1×10¹² VG of AAV-PHP.B (FIG. 24D) Representativeimage of native GFP fluorescence from the lumbar spinal cord. The insetshows an enlargement of the boxed ventral spinal cord area. (FIG. 24E)Confocal maximum projection image of GFP fluorescence from a whole mountretina. (FIG. 24F) Cross section of the retina. (FIG. 24G) CLARITYimages of GFP fluorescence from the cortex (left), striatum (right) andventral spinal cord of a mouse transduced with 1×10¹² VG of AAV-PHP.B.AAV biodistribution in the brain (FIG. 24H) and peripheral organs (FIG.24I) 25 days after injection of 1×10¹¹ VG IV into adult mice. N=3 forAAV-PHP.B and N=4 for AAV9; error bars show standard deviation (s.d.);*p<0.05, ***p<0.001, one-way ANOVA and Bonferroni's multiple comparisontest. Scale bars are 1 mm in FIG. 24A and FIG. 24D, 50 um in 24B, and200 μm in FIG. 24C. Major tick marks in FIG. 24G are 100 um.

FIGS. 25A-25J. AAV-PHP.b transduces many CNS neuronal and glial celltypes. Representative images show immunostaining for GFP (FIG. 25A-25D,FIG. 25G-251) or native GFP fluorescence (FIG. 25E, FIG. 25F, and FIG.25J). Images are of the brain regions indicated in the panel or striatum(FIG. 25E) or hippocampus (FIG. 25H). For FIGS. 25A-25F, the left imageshows the antigen immunostaining, while the right image in each pairshows GFP expression. For FIGS. 25G-25J, the top image shows theindicated antigen immunostaining, the middle images show GFP expressionand the lower paired images show a higher magnification views of theindicated boxed areas. Mice received 1×10¹¹ VG (FIG. 25A) or 1×10¹² VG(FIG. 25B-25J) at 5-6 weeks of age and were assessed for eGFP expression3 weeks later. Scale bars are 50 μm in FIG. 25A, and 20 μm in FIG.25B-25J. In all panels, arrows indicate colocalization and asterisksindicate cells that are positive for the indicated antigen but negativefor GFP.

FIGS. 26A-26E. Systemic, low-dose AAV-PHP.B reporter vector labeling canbe used together with CLARITY for single cell morphological phenotyping.(FIG. 26A) A tiled image of the cortex shows sparse labeling of cellswith GFP after IV administration of 1×10¹⁰ VG of rAAV-PHP.B:CAG-GFP. Theregion highlighted by the box is shown magnified in 3 differentorientations (FIG. 26B). Two astrocytes can be seen making contact witha blood vessel containing an endothelial cell with a GFP+ nucleus. Theastrocyte endfeet can be seen spiraling around the blood vessel. In someembodiments, this can be used to label individual cells, assess themorphology of the cells and see the material's impact on the cells.(FIG. 26C) An image of the striatum shows sparse labeling of cells withGFP after IV administration of 1×10¹⁰ VG of rAAV-PHP.B:CAG-GFP. (FIG.26D) An individual medium spiny neuron is highlighted using ansemi-automated filament tracing method (Imaris, Bitplane software).(FIG. 26E) The same medium spiny neuron highlighted in FIG. 26D is shownisolated along with several closely associated neural processes. In someembodiments, this can be used to sparsely label cells and assess theassociation of the labeled cells.

FIGS. 27A-27C Primers used for generating capsid library fragments andCre-dependent capsid sequence recovery. (FIG. 27A) Schematic shows PCRproducts as the right hand shaded section with 7AA of randomizedsequence (represented by vertical multishaded bars) inserted after aminoacid 588 (588i library) or replacing AA452-8 (452-8r library). Theprimers used to generate these libraries are indicted by name and halfarrow. For the generation of the second library, the template wasmodified to eliminate a naturally occurring EarI restriction site withinthe capsid gene fragment (xE). In this way, any contamination fromamplified wt AAV capsid sequence could be eliminated by digesting therecovered libraries with EarI. (FIG. 27B) Schematic shows therAAV-Cap-in-cis vector and the primers used to quantify vector genomes(left) and recover sequences that have transduced Cre expressing cells(left). (FIG. 27C) The figure provides the sequences for the primersshown in FIG. 27A and FIG. 27B. Table 0.1 also provides a listing of thesequences:

TABLE 0.1 Primer Purpose Sequence 9CapF Step 1: forwardCAGGTCTTCACGGACTCAGACTATCAG SEQ ID NO: 16 CDF Step 1: reversedCAAGTAAAACCTCTACAAATGTGGTAA by Cre AATCG SEQ ID NO: 17 XF Step 2 forwardACTCATCGACCAATACTTGTACTATCT CTCTAGAAC SEQ ID NO: 18 AR Step 2 reverseGGAAGTATTCCTTGGTTTTGAACCCA SEQ ID NO: 19 TF qPCR forwardGGTCGCGGTTCTTGTTTGTGGAT SEQ ID NO: 20 TR qPCR reverseGCACCTTGAAGCGCATGAACTCCT SEQ ID NO: 21 7xNNK 452-8r libraryCATCGACCAATACTTGTACTATCTCTC generation TAGAACTATTNNKNNKNNKNNKNNKNNKNNKCAAACGCTAAAATTCAGTGTGGC CGGA SEQ ID NO: 22 7xMNN 588i libraryGTATTCCTTGGTTTTGAACCCAACCGG generation TCTGCGCCTGTGCMNNMNNMNNMNNMNNMNNMNNTTGGGCACTCTGGTGGTTTG TG SEQ ID NO: 23

FIG. 27D The schematic shows an overview of some embodiments of aprocess used to introduce 2-site randomization after the first round ofselection (combinatorial libraries). This process was used to developAAV-PHP.R2. Both libraries (452-8r and 588i) were generated by PCR,cloned into the rAAV-Cap-in-cis vector and capsid selection wasperformed in TH-Cre mice. Sequences from both libraries were recoveredand were combined using an overlapping PCR strategy to generate a newlibrary that should contain all possible combinations of the 7mersequences recovered from the 452-8r library with all of the 7mersequences recovered at the 588i in one library. This library wassubjected to a second round of selection in TH-Cre mice and recoveredvariants that showed signs of enrichment were characterizedindividually.

FIG. 27E DNase-resistant vector genomes (VGs) obtained from preps ofindividual variants recovered from GFAP-Cre and TH-Cre selections.Yields are given as vector genome copies per 150 mm dish of producercells. Error bars show s.d. N=3 for AAV9, PHP.A and PHP.B. N=1 forPHP.r. One way analysis of variance (ANOVA).

FIG. 28A-28E AAV-PHP.A more efficiently and selectively transduces CNSastrocytes. (FIG. 28A) Representative images of GFP immunostaining ofbrain sections from mice injected as adults with 3×10″ VG of assAAV-CAG-eGFP expressing vector packaged into AAV9 or PHP.A asindicated. (FIG. 28B) Panels show GFP immunostaining (left) and cellnuclei (right) in the cortex of mice that received AAV9 or PHP.A asindicated. AAV biodistribution in the brain (FIG. 28C) and peripheralorgans (FIG. 28D) 25 days after injection of 1×10¹¹ VG IV into adultmice. N=4; error bars show standard deviation (s.d.); *p<0.05, **p<0.01,***p<0.001, one-way ANOVA and Bonferroni's multiple comparison test.(FIG. 28E) Representative images of GFP immunostaining of liver sectionsfrom mice injected as adults with 3×10″ VG of a ssAAV-CAG-eGFPexpressing vector packaged into AAV9 or PHP.A as indicated. Scale bar is50 μm in 28B.

FIG. 29. Rapid cellular level tropism characterization with whole animaltissue clearing. Images show native GFP fluorescence in the indicatedorgans of mice 3 weeks after IV injection of 1×10¹² AAV9 or AAV-PHP.B asindicated. The indicated organs were rendered optically transparentusing the PARS-based CLARITY (a whole body tissue clearing method—Yanget al. 2014). Confocal Z-stack images were reconstructed intothree-dimensional images using Imaris software (Bitplane).

FIG. 30 depicts a set of embodiments for amino acid and nucleic acid ofAAV9.

FIG. 31 Depicts a set of embodiments for additional targeting proteins.Any of the targeting protein embodiments provided in FIG. 31 can beswapped out for any of the embodiments involving any other particulartargeting protein embodiment described herein. Similarly, any of thenucleic acids provided in FIG. 31 can similarly be swapped out for anyof the particular nucleic acids provided herein. The figure depicts themost highly enriched sequences recovered from the second round ofselection for AAV variants that transduce GFAP-Cre+ astrocytes followingintravenous administration.

DETAILED DESCRIPTION OF EMBODIMENTS

rAAVs have reinvigorated the field of gene therapy and facilitate thegene transfer critical for a wide variety of basic science studies.Several characteristics make rAAVs attractive as gene delivery vehicles:(i) they provide long-term transgene expression, (ii) they are notassociated with any known human disease, (iii) they elicit relativelyweak immune responses, (iv) they are capable of transducing a variety ofdividing and non-dividing cell types and (v) the rAAV genome can bepackaged into a variety of capsids, or protein coat of the virus, whichhave different transduction characteristics and tissue tropisms. Despitethese advantages, the use of AAV for many applications is limited by thelack of capsid serotypes that can efficiently transduce certaindifficult cell types and by the lack of serotypes that can efficientlyand selectively target a desired cell type/organ after systemicdelivery.

Using Directed Evolution to Improve AAV Capsid Characteristics.

One approach that has been used to develop rAAVs with improvedtissue/cell type targeting is to perform directed evolution on the AAVcapsid gene. Typically this is done by making a library of replicationcompetent AAVs that are modified to introduce random mutations into theAAV cap gene, which codes for the capsid proteins that determines thetissue tropism. The AAV capsid virus library is then injected in ananimal or delivered to cells in culture. After a certain time, capsidsequences that are present in the cells/tissue of interest arerecovered. These recovered sequences are then used to generate a newpool of viruses and then the process is repeated. Through repeatedrounds of selection/sequence recovery, sequences that generate capsidsthat function better (i.e., those repeatedly pass the selection process)will be enriched. The capsids that exhibit an improved ability totransduce the target can then be recovered and assessed as individualclones or mutated further and subjected to additional rounds ofselection.

Directed evolution has been used to generate AAVs that evadeneutralizing antibodies (Maheshri et al 2006) and better target gliomacells (McGuire et al. 2010), airway epithelium (Excoffon et al. 2009)and photoreceptors in the retina after intravitreal injection (Dalkaraet al 2013). In addition, using a human/mouse chimeric liver model,Lisowski et al. developed a rAAV that specifically and efficientlytargeted the human hepatocytes (2013).

Some of the embodiments herein described provide methods for theenrichment and selective recovery of sequences with desirable traitsfrom libraries of sequence variants using a recombination-dependentrecovery strategy. This method is widely applicable for the selectiveenrichment of sequences from randomized libraries that mediate anincreased contact between the nucleic acid containing the randomizedsequence and a recombinase that recognizes a specific sequence orsequences present on the same nucleic acid as the randomized sequence.The recombinase can be expressed in response to desired stimuli, in adesired subcellular compartment or expressed in a specific targetpopulation of cells in vitro or in vivo.

As an example of the use of some embodiments, an option for selectivelyrecovering adeno-associated virus (AAV) capsid sequences that encodecapsid proteins that more efficiently and/or selectively transducespecific Cre recombinase (Cre) expressing target cell populations hasbeen provided herein. Cre recognition sites (loxP or variants of loxPsites) can be inserted into an rAAV genome adjacent or flanking to thecapsid gene. In this way, when the capsid gene enters the nucleus of aCre expressing cell and is converted to dsDNA, Cre can induce arecombination event between the lox sites within the rAAV genomeresulting on an inversion or deletion (depending on their relativeorientations) of the sequence flanked by the lox sites. Using a recoverystrategy that is dependent on the recombination event, capsid sequencesthat encode capsids that direct the rAAV genome to the nucleus of Cre+cells can be enriched through one or more rounds of selection. Capsidgene directed evolution is only one example application of thistechnology. In some embodiments, the method can be adapted for theselection of any other coding or non-coding sequences with desirabletraits within an AAV genome or any sequences within other viruses ornon-viral nucleic acids that alter the interaction with the recombinase.

The following sections provide a brief set of definitions for thevarious terms and then various embodiments that have been producedthrough these screening methods. Following this, detailed variants andembodiments of the screening method are provided, as well as examplesthereof.

Definitions

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed. In thisapplication, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise. Also, the use of the term “portion” can include partof a moiety or the entire moiety.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose. As utilized in accordance with thepresent disclosure, the following terms, unless otherwise indicated,shall be understood to have the following meanings:

A “plasmid” is as a nucleic acid that can be used to replicaterecombinant DNA sequences within a host organism. The sequence can bepreferably double stranded DNA.

The term “recombinase recognition sequence” or “recombinase recognitionsite” refers to a sequence of nucleic acid that is recognizable by arecombinase and can serve as the substrate for a recombination eventcatalyzed by said recombinase. The sequence can be double stranded DNA.

The term “virus genome” refers to a nucleic acid sequence that isflanked by cis acting nucleic acid sequences that mediate the packagingof the nucleic acid into a viral capsid. For AAVs and parvoviruses, forexample it is known that the “inverted terminal repeats” (ITRs) that arelocated at the 5′ and 3′ end of the viral genome have this function andthat the ITRs can mediate the packaging of heterologous, for example,non-wt virus genomes, into a viral capsid.

The term “element” refers to a separate or distinct part of something,for example, a nucleic acid sequence with a separate function within alonger nucleic acid sequence.

The term “rAAV” refers to a “recombinant AAV”. Recombinant AAV refers toan AAV genome in which part or all of the rep and cap genes have beenreplaced with heterologous sequences.

The term “AAV” or “adeno-associated virus” refers to a Dependoparvoviruswithin the Parvoviridae genus of viruses. Herein, AAV can refer to anAAV derived from a naturally occurring “wild-type” virus, an AAV derivedfrom a rAAV genome packaged into a capsid derived from capsid proteinsencoded by a naturally occurring cap gene and/or a rAAV genome packagedinto a capsid derived from capsid proteins encoded by a non-naturalcapsid cap gene, for example, AAV-PHP.B.

The term “rep-cap helper plasmid” refers to a plasmid that provides theviral rep and cap gene functions. This plasmid can be useful for theproduction of AAVs from rAAV genomes lacking functional rep and/or thecapsid gene sequences.

The term “vector” is defined as a vehicle for carrying or transferring anucleic acid. Examples of vectors include plasmids and viruses.

The term “cap gene” refers to the nucleic acid sequences that encodecapsid proteins that form, or contribute to the formation of, thecapsid, or protein shell, of the virus. In the case of AAV, the capsidprotein may be VP1, VP2, or VP3. For other parvoviruses, the names andnumbers of the capsid proteins can differ.

The term “rep gene” refers to the nucleic acid sequences that encode thenon-structural proteins (rep78, rep68, rep52 and rep40) required for thereplication and production of virus.

A “library” may be in the form of a multiplicity of linear nucleicacids, plasmids, viral particles or viral vectors. A library willinclude at least two linear nucleic acids.

When the inserted nucleic acid sequences are randomly generated, N=A, C,G or T; K=G or T; M=A or C.

Unless specified otherwise, the left-hand end of any single-strandedpolynucleotide sequence discussed herein is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction.

As used herein, “operably linked” means that the components to which theterm is applied are in a relationship that allows them to carry outtheir inherent functions under suitable conditions. For example, acontrol sequence in a vector that is “operably linked” to a proteincoding sequence is ligated thereto so that expression of the proteincoding sequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid sequence and therebyexpresses a gene of interest. The term includes the progeny of theparent cell, whether or not the progeny is identical in morphology or ingenetic make-up to the original parent cell, so long as the gene ofinterest is present.

The term “naturally occurring” as used herein refers to materials whichare found in nature or a form of the materials that is found in nature.

The term “treat” and “treatment” includes therapeutic treatments,prophylactic treatments, and applications in which one reduces the riskthat a subject will develop a disorder or other risk factor. Treatmentdoes not require the complete curing of a disorder and encompassesembodiments in which one reduces symptoms or underlying risk factors.

The term “prevent” does not require the 100% elimination of thepossibility of an event. Rather, it denotes that the likelihood of theoccurrence of the event has been reduced in the presence of the compoundor method.

Standard techniques can be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques can beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures can be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose. Unless specific definitions are provided, thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques can be usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients

As described herein, SEQ ID NO:4-rAAV-cap-in-cis plasmid may also bereferred to as: AAV-cap-in-cis, rAAV-Cap-in-cis vector, rAAV-CAP-in-cisgenome, rAAV-Cap-in-cis construct, rAAV9-cap-in-cis,rAAV9R-X/A-cap-in-cis, or cap-in-cis, rAAV mCherry-cap-lox71/66 genome,rAAV-CAP-in-cis-lox, AAV9 cap-in-cis genome, rAAV-cap-in-cis rAAVgenome, or the cap-in-cis rAAV genome.

As described herein, SEQ ID NO:7 (for example)—rAAV-delta-cap-in-cis mayalso be referred to as: rAAV9R-delta-X/A-cap-in-cis,rAAV9R-delta-X/A-cap-in-cis vector, rAAV-Δcap-in-cis vector,rAAV-cap-in-cis acceptor vector, cap-in-cis acceptor construct,rAAV9R-delta-X/A-cap-in-cis acceptor construct, rAAV-cap-in-cis libraryacceptor, or AAV9R-delta-X/A-cap-in-cis.

As described herein, SEQ ID NO:5 (for example)—AAV Rep-AAP helper mayalso be referred to as the Rep-AAP, rep-AAP helper and REP-AAP helper,AAV REP-AAP helper, AAV2/9 rep-AAP, or AAV2/9 REP-AAP helper plasmid.

As described herein, SEQ ID NO:6 (for example)—pCRII-9R-X/A EK plasmidor pCRII-9Cap-xE are interchangeable terms.

As described herein, AAV-PHP.B denotes the same thing as AAV-PHP.b,which denotes the same things as AAV-G2B26.

As described herein, AAV-PHP.A denotes the same things as AAV-PHP.a.

As described herein, AAV-PHP.R2 denotes the same thing as AAV-PHP.r,which denotes the same thing as AAV-TH1.1-35.

As described herein, 1253 is also referred to as 9CapF; 1316 is alsoreferred to as CDF; 1331 is also referred to as XF; 1312 is alsoreferred to as AR; 1287 is also referred to as 7×NNK; and 1286 is alsoreferred to as 7×MNN.

The term “central nervous system” or “CNS” as used herein refers to theart recognized use of the term. The CNS includes the brain, opticnerves, cranial nerves, and spinal cord. The CNS also includes thecerebrospinal fluid, which fills the ventricles of the brain and thecentral canal of the spinal cord.

Systemic administration of vectors including a capsid protein thatincludes a targeting protein of SEQ ID NO:1 of the are particularlysuitable for delivering exogenous DNA sequences encoding polypeptides,proteins, or non-coding DNA, RNA, or oligonucleotides to, for example,cells of the CNS of subjects afflicted by a CNS disease.

Targeting Sequence:

In some embodiments, a central nervous system targeting peptide isprovided. In some embodiments, the peptide comprises an amino acidsequence of SEQ ID NO: 1. In some embodiments, the peptide is furtherconjugated to a nanoparticle, a second molecule, a viral capsid protein,or inserted between amino acids 588 and 589 of AAV9 (SEQ ID NO: 2, FIG.30).

In some embodiments, the central nervous system targeting peptideincludes 4 or more amino acids of residues that overlap with residues585 to 595 within SEQ ID NO: 8.

In some embodiments, the central nervous system targeting peptideincludes 4 or more contiguous amino acids of SEQ ID NO: 1 (or any of thesequences in FIG. 31). In some embodiments, the central nervous systemtargeting peptide comprises 4-7 amino acids of SEQ ID NO: 1 (or any ofthe sequences in FIG. 31). In some embodiments, the central nervoussystem targeting peptide comprises 4-6 amino acids of SEQ ID NO: 1 (orany of the sequences in FIG. 31). In some embodiments, the centralnervous system targeting peptide includes one or more of the followingoptions: TLAVPFK (SEQ ID NO: 1); LAVPFK (SEQ ID NO: 31); AVPFK (SEQ IDNO: 32); VPFK (SEQ ID NO: 33); TLAVPF (SEQ ID NO: 34); TLAVP (SEQ ID NO:35); or TLAV (SEQ ID NO: 36). In some embodiments, the targeting peptidecan consist of, consist essentially of, or comprise one or more of thesequences in FIG. 31. In some embodiments, 2 or fewer amino acids can bealtered within TLAVPFK (or for any of the sequences within FIG. 31). Insome embodiments, one amino acid can be altered within TLAVPFK (or forany of the sequences within FIG. 31). In some embodiments, thealteration is a conservative alteration (within any of the targetingpeptides provided herein). In some embodiments, the alteration is adeletion or insertion of one or two amino acids (within any of thetargeting peptides provided herein). In some embodiments, the amino acidcan include a non-natural amino acid. In some embodiments, the centralnervous system targeting peptide sequence can be one or more of: SQTLA,QTLAV, TLAVP, LAVPK, AVPKA, VPKAQ. In some embodiments, the targetingpeptide can be at least 75% identical to one or more of the abovesequences, for example, at least 80% identical.

In some embodiments, the central nervous system targeting peptidesequence can be inverted, such as in KFPVALT (SEQ ID NO: 3) (similarly,any of the sequences in FIG. 31 can also be inverted). In suchembodiments, 4 or more contiguous amino acids can be employed. Forexample, the sequence can comprise and/or consist of KFPV, FPVA, PVAL,VALT, etc. In some embodiments, the targeting peptide can be at least75% identical to one or more of the above sequences, for example, atleast 80%.

In some embodiments, the central nervous system targeting peptidecomprises an amino acid sequence that comprises at least 4 contiguousamino acids from the sequence TLAVPFK (SEQ ID NO: 1) or KFPVALT (SEQ IDNO: 3) or any of the sequences in FIG. 31. In some embodiments, theamino acid sequence results in an increase in CNS cell transduction bythe AAV. In some embodiments, the amino acid sequence is part of acapsid protein of the AAV vector. In some embodiments, the sequenceTLAVPFK (SEQ ID NO: 1; (or any of the sequences in FIG. 31)) is insertedbetween AA588-589 of an AAV sequence of the vector (SEQ ID NO: 2). Insome embodiments, the sequence TLAVPFK (SEQ ID NO: 1; or any of thesequences in FIG. 31) is inserted between AA586-592 of an AAV sequenceof the vector (SEQ ID NO: 2). In some embodiments, the sequence TLAVPFK(SEQ ID NO: 1; or any of the sequences in FIG. 31) further comprises atleast two of amino acids 587, 588, 589, or 590 of SEQ ID NO: 2. In someembodiments, the targeting peptide can be at least 75% identical to oneor more of the above sequences.

In some embodiments, the central nervous system targeting peptidecomprises, consists, or consists essentially of any one or more of theabove sequences. In some embodiments, the central nervous systemtargeting peptide is inserted into a longer peptide, as describedherein.

In some embodiments, the targeting peptide is part of an AAV, asdescribed herein. In some embodiments, the targeting peptide is part ofan AAV9. In some embodiments, the targeting peptide can be linked to anymolecule that should be targeted as desired. In some embodiments, thetargeting peptide can be linked, without limitation, to a recombinantprotein, antibody, a cell, a diagnostic, a therapeutic, a nanomolecule,etc.

In some embodiments, the targeting sequence is an amino acid sequence.In some embodiments, the targeting sequence is a nucleic acid sequence.In some embodiments, the targeting sequence is a capsid proteincomprising an amino acid sequence that comprises at least 4 contiguousamino acids from the sequence TLAVPFK (SEQ ID NO: 1) and/or KFPVALT (SEQID NO: 3) and/or any of the sequences in FIG. 31.

Some embodiments of options of targeting sequences, as outlined in theexamples below, are provided in FIG. 1A, which includes G2B-13, G2B-26,TH1.1-32, and TH1.1-35, by way of example.

In some embodiments, the targeting protein can be inserted into anydesired section of a protein. In some embodiments, the targeting proteincan be inserted into a capsid protein. In some embodiments, thetargeting protein is inserted on a surface of the desired protein. Insome embodiments, the targeting protein is inserted into the primarysequence of the protein. In some embodiments, the targeting protein islinked to the protein. In some embodiments, the targeting protein iscovalently linked to the protein. In some embodiments, the targetingprotein is inserted into an unstructured loop of the desired protein. Insome embodiments, the unstructured loop can be one identified via astructural model of the protein.

In some embodiments, the unstructured loop can be one identified bysequence comparisons, such as shown in FIG. 13. FIG. 13 shows analignment of VP1 capsid amino sequence from AAV and related parvovirusesaligned to AAV9. Sequence identity is shown as a dot. The AAs thatdiffer from AAV9 are indicated. The numbering is based on AAV9 VP1. OnlyAA 418-624 are shown, although such an alignment can be done by one ofskill in the art for any desired section of protein. Shaded verticalbars of different length represent the relative conservation at each AA.Longer bars indicate greater conservation. Horizontal shaded barsindicate sites of the unstructured loops into which the targetingprotein can be inserted.

In some embodiments, the location of insertion of the targeting proteininto the desired protein can be achieved by a structural model. Anexample of such a structural model is shown in FIG. 14. FIG. 14 depictsa structural model highlighting surface loops randomized by targetedsequence insertion. The insert (which is a blow up) depicts a ribbondiagram of AAV9 surface model constructed in PyMol from the AAV9 ProteinData Bank file 3ux1.pdb. The capsid surface is shown in gray and theloop regions chosen for sequence insertion are highlighted by shading(AA586-592) and (AA452-458). Other regions of sequence insertion orreplacement can be identified from within regions that are not highlyconserved. Additional examples include the regions of AAV9 betweenAA262-269, AA464-473, AA491-495, AA546-557 and AA659-668 or thehomologous regions of other the capsid proteins from other AAVs orparvoviruses.

In some embodiments, the capsid protein can comprise or consist of thesequence shown in FIG. 19, SEQ ID NO: 8. The underlined amino acid is aK to R mutation that was made to provide a unique XbaI restriction site,this can be optional. For references, SEQ ID NO: 1 (TLAVPFK) is in boldtext. Any of the other targeting peptides provided herein (for example,in FIG. 31) can also be inserted in place of SEQ ID NO: 1. FIG. 20depicts some embodiments of a nucleic acid sequence for an AAV-PHP.B(AAV-G2B26) capsid gene coding sequence). The recovered nucleic acidsequence encoding SEQ ID NO: 1 (TLAVPFK) is in bold and underlined text.The mutations introduced to insert or remove restriction sites arehighlighted with double underlined italicized text.

Vectors

In some embodiments, a viral vector can include one or more of the notedtargeting sequences (for example, any of the central nervous systemtargeting peptides noted herein or any peptide provided by the screeningmethods provided herein). In some embodiments, an AAV vector can beprovided that comprises a sequence TLAVPFK (SEQ ID NO: 1) (or any of theother targeting proteins provided herein, including those in FIG. 31).

In some embodiments, one or more targeting sequences can be employed ina single system. For example one can employ one or more targetingsequences and also modify other sites to reduce the recognition of theAAVs by the pre-existing antibodies present in the host, such as ahuman. In some embodiments, the AAV vector can include a capsid, whichinfluences the tropism/targeting, speed of expression and possibleimmune response. The vector can also include the rAAV, which genomecarries the transgene/therapeutic aspects (e.g., sequences) along withregulatory sequences. In some embodiments, the vector can include thetargeting sequence within/on a substrate that is or transports thedesired molecule (therapeutic molecule, diagnostic molecule, etc.).

In some embodiments, any one or more of the targeting sequences providedherein can be incorporated into a vector.

In some embodiments, the sequence TLAVPFK (SEQ ID NO: 1) (or any of theother targeting proteins provided herein, including those in FIG. 31)results in an increase in CNS cell transduction from a virus containingthe vector. In some embodiments, the increase is at least 2, 10, 20, 30,40, 50, 60, 70, 80, 90, or at least 100 fold more than transductionwithout the targeting sequence. In some embodiments, there is a 40-90fold increase in transduction of the CNS, as compared with AAV9transduction.

In some embodiments, the sequence TLAVPFK (SEQ ID NO: 1) (or any of theother targeting proteins provided herein, including those in FIG. 31) ispart of a capsid protein of the AAV vector. In some embodiments, thesequence TLAVPFK (SEQ ID NO: 1) (or any of the other targeting proteinsprovided herein, including those in FIG. 31) is inserted betweenAA588-589 of an AAV sequence of the vector (SEQ ID NO: 2). In someembodiments, the sequence TLAVPFK (SEQ ID NO:1) (or any of the othertargeting proteins provided herein, including those in FIG. 31) isinserted within AA452-458 of an AAV sequence of the vector (SEQ ID NO:2). In some embodiments, the sequence TLAVPFK (SEQ ID NO:1) (or any ofthe other targeting proteins provided herein, including those in FIG.31) is inserted within AA491-495 of an AAV sequence of the vector (SEQID NO: 2). In some embodiments, the sequence TLAVPFK (SEQ ID NO:1) (orany of the other targeting proteins provided herein, including those inFIG. 31) is inserted within AA546-557 of an AAV sequence of the vector(SEQ ID NO: 2). In some embodiments, any of the targeting sequences (orcombination thereof) in FIG. 31 can be used and/or substituted for anyof the embodiments provided herein regarding SEQ ID NO: 1. Thus, forexample, one or more of the sequences within FIG. 31 can be insertedbetween AA588-589 of an AAV sequence of the vector (SEQ ID NO: 2). Insome embodiments, the targeting sequence can be one or more of: SVSKPFL(SEQ ID NO: 28); FTLTTPK (SEQ ID NO: 29); or MNATKNV (SEQ ID NO: 30).FIG. 31 depicts some of the most highly enriched sequences recoveredfrom the second round of selection for AAV variants that transduceGFAP-Cre+ astrocytes following intravenous administration.

In some embodiments, the targeting sequence that is part of the vectorcan comprise any four contiguous AAs within AAV9 VP1 AA585-598 of SEQ IDNO: 8.

While the numbering is not identical between serotypes, the exactinsertion site is not critical. In some embodiments, the targetingsequence is inserted within the unstructured (see FIG. 14) and poorlyconserved (see alignment, FIG. 13) surface exposed loops. In someembodiments, the insertion of the targeting sequence can be achievedwithin other AAV capsids by inserting the targeting sequence within thehomologous unstructured loops of other AAV sequences.

In some embodiments, an rAAV genome is provided. The genome can compriseat least one inverted terminal repeat configured to allow packaging intoa vector and a cap gene. In some embodiments, it can further include asequence within a rep gene required for expression and splicing of thecap gene. In some embodiments, the genome can further include a sequencecapable of expressing VP3.

In some embodiments, the only protein that is expressed is VP3 (thesmallest of the capsid structural proteins that makes up most of theassembled capsid—the assembled capsid is composed of 60 units of VPproteins, ˜50 of which are VP3). In some embodiments, VP3 expressionalone is adequate to allow the method of screening to be adequate.

In some embodiments, the system for screening involves placing theselectable element, (which in some embodiments can be the AAV cap geneinto the AAV genome) together with one or more recombinase recognitionsites (loxP or mutant loxP sites are preferred, but others could beused). In some embodiments, the AAV genome can be defined by a nucleicacid comprising at least one inverted terminal repeat.

In some embodiments, the rAAV genome further comprises a mCherryreporter cassette comprising a ubiquitin C gene, a mCherry cDNA, and aminimal synthetic polyA sequence.

In some embodiments, the genome further comprises cre-dependent switchcomprising: a polyA sequence and a pair of inverted loxP sites flankingthe polyA sequence. In some embodiments, the polyA sequence isdownstream of the cap gene. In some embodiments, the pair of invertedloxP sites comprises lox71 and lox66. In some embodiments, the genomecontains only those sequences within the rep gene required forexpression and splicing of the cap gene product.

In some embodiments, AAV-PHP.B delivers genes efficiently to one or moreorgans including, but not limited to the central nervous system, liver,muscle, heart, lungs, stomach, adrenal gland, adipose and intestine.

In some embodiments, a capsid library is provided that comprises AAVgenomes that contain both the full rep and cap sequence that have beenmodified so as to not prevent the replication of the virus underconditions in which it could normally replicate (co-infection of amammalian cell along with a helper virus such as adenovirus). A pseudowt genome can be one that has an engineered cap gene within a “wt” AAVgenome.

In some embodiments, the capsid library is made within a “pseudo-wildtype” AAV genome containing the viral replication gene (rep) and capsidgene (cap) flanked by inverted terminal repeats (ITRs). In someembodiments, the capsid library is not made within a “pseudo-wild type”AAV genome containing the viral replication gene (rep) and capsid gene(cap) flanked by inverted terminal repeats (ITRs).

In some embodiments, the rAAV genome contains the cap gene and onlythose sequences within the rep gene required for the expression andsplicing of the cap gene products (FIG. 22B).

In some embodiments, a capsid gene recombinase recognition sequence isprovided with inverted terminal repeats flanking these sequences.

In some embodiments, the system could be used to develop capsids thatexhibit enhanced targeting of specific cells/organs, select for capsidsthat evade immunity, select for genomes that are more at homologousrecombination, select for genome elements that increase the efficiencyof conversion of the single stranded AAV genome to a double stranded DNAgenome within a cell and/or select for genome elements that increase theconversion of AAV genome to a persistent, circularized form within thecell.

Nucleic Acid Sequences

In some embodiments, a nucleic acid sequence encoding any of thetargeting sequences provided herein is provided. In some embodiments,the nucleic acid sequence is AAGTTTCCTGTGGCGTTGACT FOR SEQ ID NO 3). ACTTTG GCG GTG CCT TTT AAG (SEQ ID NO:49) for a sequence encoding the AAsequence of SEQ ID NO: 1. In some embodiments, the nucleic acid sequenceis one that will hybridize to this sequence under stringent conditions.In some embodiments, the nucleic acid sequence includes a nucleic acidsequence that encodes for SEQ ID NOS: 1 and/or 3 and the sequence ispart of a larger nucleic acid sequence. In some embodiments, any one ormore of the sequences from FIG. 31 can provide the noted nucleic acidsequence (that is, any nucleic acid sequence that encodes for any ofthese sequences can be provided). In some embodiments, the nucleic acidsequence is one that will hybridize to any of the sequences within FIG.31 (or the sequences that encode the amino acid sequences) understringent conditions. In some embodiments, the nucleic acid sequenceincludes a nucleic acid sequence that encodes for any of the sequenceswithin FIG. 31 and the sequence is part of a larger nucleic acidsequence. In some embodiments, the nucleic acid sequence is one or moreof:

(SEQ ID NO: 24) AGTGTGAGTAAGCCTTTTTTG; (SEQ ID NO: 26)TTTACGTTGACGACGCCTAAG; or (SEQ ID NO: 27) ATGAATGCTACGAAGAATGTG.

In some embodiments, the nucleic acid sequence that encodes for SEQ IDNO: 1 is inserted between a sequence encoding for amino acids 588 and589 of AAV9 (SEQ ID NO: 2).

In some embodiments, a nucleic acid sequence encoding any fourcontiguous amino acids in TLAVPFK (SEQ ID NO: 1) or in KFPVALT (SEQ IDNO: 3) is provided. In some embodiments, a nucleic acid sequenceencoding any five contiguous amino acids in TLAVPFK (SEQ ID NO: 1) or inKFPVALT (SEQ ID NO: 3) is provided. In some embodiments, a nucleic acidsequence encoding any six contiguous amino acids in TLAVPFK (SEQ IDNO: 1) or in KFPVALT (SEQ ID NO: 3) (or any of the other targetingproteins provided herein, including those in FIG. 31) is provided.

In some embodiments, the nucleic acid sequence is inserted between asequence encoding for amino acids 588 and 589 of AAV9 (SEQ ID NO: 2).

In some embodiments, a plasmid system is provided. The plasmid caninclude a first plasmid comprising a modified AAV2/9 rep-cap helperplasmid comprising at least one in frame stop codon within its VP1, VP2and VP3 reading frame. The stop codon is positioned to disrupt VPexpression without altering the amino acid sequence of the assemblyactivating protein. The plasmid system can further include a secondplasmid comprising a rAAV-cap-in-cis plasmid.

In some embodiments, the method does not involve expressing single VPproteins from heterologous plasmids to generate “mosaic” capsids madefrom VP proteins encoded by different plasmids.

In some embodiments, a library of nucleic acid sequences is provided.The library can comprise a selectable element and one or morerecombinase recognition sequences. In some embodiments, the nucleic acidsequences and one or more recombinase recognition sequences areincorporated within a virus genome. In some embodiments, the viralgenome is an AAV genome. In some embodiments, the selectable elementencodes an AAV capsid. In some embodiments, the selectable element is agenetic element that increases conversion to dsDNA. In some embodiments,the selectable element increases the efficiency of homologousrecombination between the element and the endogenous genome. In someembodiments, the recombinase recognition sequences are comprised of oneor more loxP sites. In some embodiments, the loxP site is a lox71 siteand an inverted lox66 site.

In some embodiments, the gene encoding the targeting protein and/or thecapsid can be cloned into an AAV Rep-Cap helper plasmid in place of theexisting capsid gene. When introduced together into producer cells, thisplasmid can be used to package an rAAV genome into the targeting proteinand/or capsid. Producer cells can be any cell type possessing the genesnecessary to promote AAV genome replication, capsid assembly andpackaging. Preferred producer cells are 293 cells, or derivatives, HELAcells or insect cells together with helper virus or a second plasmidencoding the helper virus genes known to promote rAAV genomereplication. In some embodiments, an AAV rep-cap helper sequence can bemodified to introduce a tetracycline-inducible expression system inbetween the rep and the cap gene to increase capsid expression and virusproduction. In some embodiments, a tetracycline transactivator cDNA,poly adenylation sequence, tetracycline responsive element and AAV5 p41promoter and AAV2 splicing regulatory elements contained within the AAV2rep gene are inserted between the rep gene and the gene encoding thecapsid or targeting protein. Use of this inducible rep-cap plasmid whenmaking rAAV provides 1.5-2-fold more virus than the AAV2/9 rep-capplasmid. Some embodiments of such a nucleic acid cloned into a plasmidare depicted in FIG. 21, SEQ ID NO: 10. The cap gene sequence isunderlined in FIG. 21. Uppercase letters indicate sites where the capsidsequence differs from AAV9.

Methods of Use

In some embodiments, a method of delivering a nucleic acid sequence to anervous system (or other desired system) is provided. The method caninclude providing a protein comprising any one or more of the targetingsequences provided herein. The protein can be part of a capsid of anAAV. The AAV can comprise a nucleic acid sequence to be delivered to anervous system. One can then administer the AAV to the subject.

In some embodiments, the nucleic acid sequence to be delivered to anervous system comprises one or more sequences that would be of some useor benefit to the nervous system and/or the local of delivery orsurrounding tissue or environment. In some embodiments, it can be anucleic acid that encodes a trophic factor, a growth factor, or othersoluble factors that might be released from the transduced cells andaffect the survival or function of that cell and/or surrounding cells.In some embodiments, it can be a cDNA that restores protein function tohumans or animals harboring a genetic mutation(s) in that gene. In someembodiments, it can be a cDNA that encodes a protein that can be used tocontrol or alter the activity or state of a cell. In some embodiments,it can be a cDNA that encodes a protein or a nucleic acid used forassessing the state of a cell. In some embodiments, it can be a cDNAand/or associated RNA for performing genomic engineering. In someembodiments, it can be a sequence for genome editing via homologousrecombination. In some embodiments, it can be a DNA sequence encoding atherapeutic RNA. In some embodiments, it can be a shRNA or an artificialmiRNA delivery system. In some embodiments, it can be a DNA sequencethat influences the splicing of an endogenous gene.

In some embodiments, the resulting targeting molecules can be employedin methods and/or therapies relating to in vivo gene transferapplications to long-lived cell populations. In some embodiments, thesecan be applied to any rAAV-based gene therapy, including, for example:spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS),Parkinson's disease, Pompe disease, Huntington's disease, Alzheimer'sdisease, Battens disease, lysosomal storage disorders, glioblastomamultiforme, Rett syndrome, Leber's congenital amaurosis, chronic pain,stroke, spinal cord injury, traumatic brain injury and lysosomal storagedisorders. In addition, rAAVs can also be employed for in vivo deliveryof transgenes for non-therapeutic scientific studies such asoptogenetics, gene overexpression, gene knock-down with shRNA or miRNAs,modulation of endogenous miRNAs using miRNA sponges or decoys,recombinase delivery for conditional gene deletion, conditional(recombinase-dependent) expression, or gene editing with CRISPRs,TALENs, and zinc finger nucleases.

Provided herein are methods for treating and/or preventing Huntington'sdisease using the methods and compositions described herein. The methodof treating and/or preventing Huntington's disease can includeidentifying the subject(s), providing a vector for delivery of apolynucleotide to the nervous system of the subject as provided herein,administering the vector in an effective dose to the subject therebytreating and/or preventing Huntington's disease in the subject. In someembodiments, the methods for treating a subject with Huntington'sdisease involve compositions where the vector delivers thepolynucleotide composition comprising a Zinc finger protein (ZFP)engineered to represses the transcription of the Huntingtin (HTT) gene.In some embodiments, the ZFP selectively represses the transcription ofthe HTT gene allele responsible for causing the Huntington's disease inthe subject by binding to the CAG repeat region of the HTT gene in a CAGrepeat length-dependent manner. In some embodiments, the ZNFTRselectively represses transcription of both alleles of the HTT gene.

In some embodiments, the therapeutic item to be administered to thesubject comprises a short hairpin RNA (shRNA) or microRNA (miRNA) thatknocks down Huntingtin expression by inducing the selective degradationof, or inhibiting translation from, RNA molecules transcribed from thedisease causing HTT allele by binding to the CAG repeat. In someembodiments, the therapeutic item to be administered to the subjectcomprises a short hairpin RNA (shRNA) or microRNA (miRNA) that knocksdown Huntingtin expression by inducing the degradation of, or inhibitingtranslation from, RNA molecules transcribed from one or both alleles ofthe HTT gene. In some embodiments, the therapeutic item to beadministered to the subject comprises a short hairpin RNA (shRNA) ormicroRNA (miRNA) that knocks down Huntingtin expression by inducing theselective degradation of, or inhibiting translation from, RNA moleculestranscribed from the disease causing HTT allele through the selectiverecognition of one or more nucleotide polymorphisms present within thedisease causing allele. The nucleotide polymorphisms can be used by oneskilled in the art to differentiate between the normal and diseasecausing allele.

In some embodiments, the therapeutic item to be administered to thesubject comprises a polynucleotide that encodes an RNA or protein thatalters the splicing or production of the HTT RNA. In some embodiments,the therapeutic item to be administered to the subject comprises apolynucleotide that encodes one or more polypeptides and/or RNAs forgenome editing using a Transcription activator-like effector nuclease(TALEN), zinc finger nuclease or clustered regularly interspaced shortpalindromic repeats—cas9 gene (CRISPR/Cap9) system engineered by oneskilled in the art to induce a DNA nick or double-stranded DNA breakwithin or adjacent to the HTT gene to cause an alteration in the HTTgene sequence. In some embodiments, the therapeutic item to beadministered to the subject comprises a polynucleotide encoding apolypeptide that binds to a polypeptide from the HTT gene, alters theconformation of a polypeptide from the HTT gene or alters the assemblyof a polypeptide from the HTT gene into aggregates or alters thehalf-life of a polypeptide from the HTT gene. In some embodiments, thetherapeutic item to be administered to the subject comprises apolynucleotide that encodes a RNA or polypeptide that causes or preventsa post-transcriptional modification of a polypeptide from the HTT gene.In some embodiments, the therapeutic item to be administered to thesubject comprises a polynucleotide that encodes a polypeptide from achaperone protein known to those skilled in the art to influence theconformation and/or stability of a polypeptide from the HTT gene.

In some embodiments, the therapeutic item to be administered to thesubject comprises regulatory elements known to one skilled in the art toinfluence the expression of the RNA and/or protein products encoded bythe polynucleotide within desired cells of the subject.

In some embodiments, the therapeutic item to be administered to thesubject comprises a therapeutic item applicable for any disease ordisorder of choice. In some embodiments, this can include compositionsfor treating and/or preventing Alzheimers disease using the methods andcompositions described herein, for example, ApoE2 or ApoE3 forAlzheimer's disease; SMN for the treatment of SMA; frataxin delivery forthe treatment of Friedreich's ataxia; and/or shRNA or miRNA for thetreatment of ALS.

In some embodiments, the therapeutic item for delivery is a protein(encodes a protein) or RNA based strategy for reducing synucleinaggregation for the treatment of Parkinson's. For example delivering apolynucleotide that encodes a synuclein variant that is resistant toaggregation and thus disrupts the aggregation of the endogenoussynuclein.

In some embodiments, a transgene encoding a trophic factor for thetreatment of AD, PD, ALS, SMA, HD can be the therapeutic item involved.In some embodiments, a trophic factor can be employed and can include,for example, BDNF, GDNF, NGF, LIF, and/or CNTF.

Dosages of a viral vector can depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectivehuman dosage of the viral vector is generally in the range of from about0.1 ml to about 100 ml of solution containing concentrations of fromabout 1×10⁹ to 1×10¹⁶ genomes virus vector. A preferred human dosage canbe about 1×10¹³ to 1×10¹⁶ AAV genomes. The dosage will be adjusted tobalance the therapeutic benefit against any side effects and suchdosages may vary depending upon the therapeutic application for whichthe recombinant vector is employed. The levels of expression of thetransgene can be monitored to determine the frequency of dosageresulting from the vector of the invention.

In some embodiments, the polynucleotides vector also includes regulatorycontrol elements known to one skilled in the art to influence theexpression of the RNA and/or protein products encoded by thepolynucleotide within desired cells of the subject.

In some embodiments, functionally, expression of the polynucleotide isat least in part controllable by the operably linked regulatory elementssuch that the element(s) modulates transcription of the polynucleotide,transport, processing and stability of the RNA encoded by thepolynucleotide and, as appropriate, translation of the transcript. Aspecific example of an expression control element is a promoter, whichis usually located 5′ of the transcribed sequence. Another example of anexpression control element is an enhancer, which can be located 5′ or 3′of the transcribed sequence, or within the transcribed sequence. Anotherexample of a regulatory element is a recognition sequence for amicroRNA. Another example of a regulatory element is an intron and thesplice donor and splice acceptor sequences that regulate the splicing ofsaid intron. Another example of a regulatory element is a transcriptiontermination signal and/or a polyadenylation sequences.

Expression control elements and promoters include those active in aparticular tissue or cell type, referred to herein as a “tissue-specificexpression control elements/promoters.” Tissue-specific expressioncontrol elements are typically active in specific cell or tissue (forexample in the liver, brain, central nervous system, spinal cord, eye,retina or lung). Expression control elements are typically active inthese cells, tissues or organs because they are recognized bytranscriptional activator proteins, or other regulators oftranscription, that are unique to a specific cell, tissue or organ type.

Expression control elements also include ubiquitous or promiscuouspromoters/enhancers which are capable of driving expression of apolynucleotide in many different cell types. Such elements include, butare not limited to the cytomegalovirus (CMV) immediate earlypromoter/enhancer sequences, the Rous sarcoma virus (RSV)promoter/enhancer sequences and the other viral promoters/enhancersactive in a variety of mammalian cell types; promoter/enhancer sequencesfrom ubiquitously or promiscuously expressed mammalian genes including,but not limited to, beta actin, ubiquitin or EF1alpha; or syntheticelements that are not present in nature.

Expression control elements also can confer expression in a manner thatis regulatable, that is, a signal or stimuli increases or decreasesexpression of the operably linked polynucleotide. A regulatable elementthat increases expression of the operably linked polynucleotide inresponse to a signal or stimuli is also referred to as an “inducibleelement” (that is, it is induced by a signal). Particular examplesinclude, but are not limited to, a hormone (for example; steroid)inducible promoter. A regulatable element that decreases expression ofthe operably linked polynucleotide in response to a signal or stimuli isreferred to as a “repressible element” (that is, the signal decreasesexpression such that when the signal, is removed or absent, expressionis increased). Typically, the amount of increase or decrease conferredby such elements is proportional to the amount of signal or stimulipresent; the greater the amount of signal or stimuli, the greater theincrease or decrease in expression.

Any one or more of the above aspects can be included within any of thevectors provided herein, in combination with any targeting protein.

Method of Selection

Current directed evolution protocols used to enhance AAV capsids haveseveral shortcomings. The first is that it is difficult to design an invivo screen that specifically recovers sequences from the target cell ofinterest when that target cell is one of many cell types in a complexorgan. Typically, after the virus is administered in vivo, the tissue ofinterest is collected, and virus DNA is recovered from the DNA of theentire tissue, or region of tissue. Recently, Dalkara et al. reportedthe use of FACS sorting the target cells (photoreceptors) as a means toselectively recover capsid sequences present in those cells. But thismethod is labor intensive and costly, especially for sorting from largevolumes of dissociated tissues. In addition, this additional sortingeffort does not overcome the other major limitation of selecting for AAVcapsid sequences: all capsid sequences present within the cell/tissueare recovered regardless of whether or not these viruses functionallytransduced any cells. In other words, sequences from viruses stuck onthe cell surface or viruses that entered the cell bound to a receptorthat trafficked to an intracellular compartment not compatible with AAVunpackaging and transduction are recovered by these screens along withsequences that encoded capsids that successfully induced transgeneexpression in the target cell population. Therefore, non-functionalcapsids are also enriched by typical selection methods. Finally, mostcurrent methods also require the use of libraries made from replicationcompetent AAV, which is a potential biosafety concern, especially if thevirus will be introduced in animal facilities where there are primatessince these viruses could replicate in animals carrying helper viruses.Herein is described an AAV capsid library screening platform thatovercomes one or more of each of these limitations.

Successful production of an AAV capsid variant library depends upon eachvariant cap gene being packaged by the particular capsid proteins itencodes. Therefore, it is useful that the cap gene is present in cis(within the AAV genome). However, it is not essential that thenon-structural rep genes be present in cis. Herein is disclosed areplication incompetent rAAV genome expressing the cap gene and in placeof much of the rep sequence, several recombinant elements have beenadded that provide a way to selectively recover only those capsidsequences that have functionally transduced the target cell populationof interest without the need for target cell isolation.

Selective Recovery of AAV Capsid Sequences from Specific Cre+ CellPopulations

In some embodiments, the approach incorporates a Crerecombinase-dependent switch that uses PCR (polymerase chain reaction)to selectively recover capsid sequences that have transduced Cre+ targetcells. This can be accomplished by inserting mutant Cre recognitionsites (lox66 and lox71 in a head-to-head orientation) into the rAAVgenome around a sequence adjacent to the cap gene (FIG. 1B). Crerecombination results in an inversion of the sequence flanked by thelox66 and lox71 sites, and one can then use a PCR recovery strategy thatonly amplifies the cap gene sequence from rAAV genomes afterCre-mediated inversion of the cap gene adjacent sequence (one PCR primerbinds the invertible sequence and one binds the cap gene). Mutant loxPsites (lox66 and lox71) can be chosen so that the inversion would beless reversible (Alberts et al. 1995). FIG. 1B shows an embodiments of arAAV plasmid that has been developed.

In some embodiments, the method takes advantage of the large number ofCre transgenic mice that have been (and can be) developed. These linesexpress Cre under the control of cell specific promoters such that Creis present only in a subpopulation of cells within a given organ.Hundreds of Cre transgenic lines are available from commercial vendorsand academic sources, and custom lines can be generated.

In some embodiments, one can apply this for developing capsids that moreefficiently transduce astrocytes in the central nervous system after IVvirus administration. Transgenic mGFAP-Cre mice are available thatexpress Cre specifically within astrocytes and neural stem cells (NSCs)in the adult brain and spinal cord. Using the Cre-dependent sequencerecovery strategy, one can deliver rAAV capsid libraries in vivo,collect DNA from the entire brain and spinal cord and recover rAAVcapsid sequences specifically from astrocytes and NSCs.

Another advantage of some embodiments of this approach is that this Credependent strategy only recovers those sequences that have transducedthe target cell. AAV is a single stranded DNA virus, and its genome mustenter the nucleus and be converted to double stranded DNA (dsDNA) forfunctional transduction. Since Cre only recombines dsDNA, only thosecapsid sequences that have trafficked properly to the cell nucleus andhave been converted to dsDNA will be recovered.

Inclusion of a Reporter Gene Cassette to Facilitate Cell Sorting

For cases where Cre+ transgenics are not available, one can alsoincorporate a reporter cassette driven by a ubiquitous promoter/enhancerto facilitate sorting of transduced/transgene expressing cells fromwithin a mixed population. This second option is more labor intensivethan the Cre-based strategy as it requires generating single cellsuspensions and FACS or magnetic bead/antibody-based sorting. But thereporter method is also powerful in that it can be combined with sortingfor specific target cell populations using antibodies to known surfacemarkers or with GFP transgenics to limit recovery to a particularpopulation. And like the Cre strategy above, it will only lead to therecovery of sequences that are present in transduced cells. The reporteralso facilitates following the transduction characteristics of thepooled library during screening (useful for both the Cre- andreporter-dependent methods).

The technology described herein can be used in conjunction with anytransgenic line expressing Cre in the target cell type of interest todevelop AAV capsids that more efficiently transduce that target cellpopulation. Applications include, but are not limited to, developingcapsids that are more efficient at transducing specific cell types inany organ after IV AAV administration, targeting specific populations ofneurons, improving interneuronal transport, targeting tumor cells,hematopoetic stem cells, insulin producing beta cells, lung epithelium,etc. The method is not limited to any one virus delivery method. Thevector may be delivered via any route including, but not limited to,oral, intravenous, intraarticular, intracardiac, intramuscular,intradermal, topical, intranasal, intraparitoneal, rectal, sublingual,subcutaneous, epidural, intracerebral, intracerebroventricular,intrathecal, intravitreal or subretinal administrations. The system canalso be used to develop viruses that better cross specific barriers(blood brain barrier, gut epithelium, placenta, etc.). The method canalso be used in vitro to develop capsids that are better at achievingnuclear entry and second strand synthesis (conversion to dsDNA).

In addition, this system is not limited to AAV9. Any starting AAV capsid(naturally occurring or modified variants) can be incorporated into thisrAAV-cap vector, mutagenized by standard methods to create the capsidlibrary and then screened with this Cre-dependent recovery strategy.Preferred AAV capsids include AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, human isolates (for example, hu.31 and hu.32),rhesus isolates (for example, rh.8 and rh.10), AAVs or relatedparvoviruses from other primates, mammals and non-mammalian species.Furthermore, this method is not limited to any one commonly used capsidmutagenesis strategy. Any method can be used to generate the librarydiversity, including but not limited to capsid domain shuffling, randomsequence insertion and random mutagenesis by error prone PCR. Finally,the vector has been designed to be modular making it simple to replacevarious elements such as the reporter cassette or capsid sequence tofurther customize the screening options.

To make this system possible, an option for incorporating the AAV capsidgene within the rAAV genome while providing the other non-structural AAVgene products in trans from a helper plasmid was developed. This was notnecessarily straightforward since the cap gene codes for 4 proteins (thecapsid proteins VP1, VP2, VP3 and the assembly activating protein (AAP))using a combination of alternative splicing, alternative start codonsand alternative reading frames. Maintaining proper regulation of theexpression of these proteins is relevant for efficient virus production.It was found that the capsid proteins could be expressed from a rAAVgenome when a fragment of the 3′ end of the rep gene, which containscritical promoter/enhancer and splicing signals, is included (FIG. 2A).Retaining only the 3′ end of the rep gene together with the capsid geneleft enough space within the rAAV genome to incorporate the Creinvertible polyA sequence downstream of the cap gene as well as anmCherry (red fluorescent protein) reporter cassette.

To insure that the capsid is made entirely from the capsid gene encodedwithin the rAAA capsid library genome, an AAV helper plasmid that wouldprovide the AAV non-structural proteins but not any capsid proteinexpression (typically rAAV are produced by supplying both rep and capgenes in trans from the AAV helper plasmid) was developed. Using aAAV2/9 RepCap plasmid vector core as a starting point, 5 stop codonswere inserted within the cap gene near the translation initiation sitesfor the three capsid proteins VP1, VP2 and VP3 (FIG. 2B). Thiseffectively eliminated rAAV production unless the VP1-3 capsid proteinsare provided elsewhere (such as in cis on the AAV genome cap libraryconstruct described above).

In some embodiments, a method of developing a capsid with a desiredcharacteristic is provided. The method can comprise providing apopulation of rAAV genomes provided herein. The method can furtherinvolve screening the population by a specific set of criteria. Themethod can further involve selecting the rAVV genome that meets thescreening criteria.

In some embodiments, some of the methods of screening provided hereinprovide at least one of the following advantages. First, in someembodiments, the method makes use of the growing library ofCre-transgenics to provide selective pressure for capsids that moreefficiently transduce genetically defined cell populations (for example,see cre.jax, gensat, creportal, connectivity.brain-map (MGI) (all“.org”). Second, in some embodiments, since Cre only recombines doublestranded DNA (dsDNA) and the AAV genome is single stranded, only thosecapsid sequences that mediate the proper intracellular trafficking andconversion of the packaged genome to a persistent dsDNA form will berecovered. Therefore, such an approach can provide additional selectivepressure for functional capsids.

As depicted in FIG. 2C, additional sequence variants can be selectedbased on their ability to mediated an increased association of thenucleic acid carrying the sequence library and recombinase site with therecombinase for similar or different desired applications. In someembodiments, the process can start by generating a library of DNAsequence variants (1A). In some embodiments, this can include 10̂2 if notmore sequences (for example 10̂6 or more). Within the same nucleic acid,one can also incorporate one or more recombinase recognition sequences(1B). A strategy is then designed for recombination-dependent sequencerecovery/amplification of the sequence variants. (1B). This can involveone or more recombinase recognition sites. One can then combine thelibrary and recombination sites to transfer library DNA fragments into avector with recombinase recognition sites (2). One can then deliver thelibrary (for example, in vivo and/or in vitro). The recombinase (REC+)expression is restricted to one or more target cell populations orcompartments (3). One can then apply a selected selective pressure tothe system such that one can recover/amplify sequences based on thepresence or absence of the recombinase-mediated recombination events onthe nucleic acids comprising the library variants (4). This process canbe repeated if necessary, transferring the recovered or amplifiedselected variants back into the library acceptor vector (5B) for 1 to 5,or more rounds of selection. One can then obtain and characterize thevariant sequences (5A) by various methods, such as Sanger sequencing ornext generation sequencing. Finally, one can then characterize thefunction of any or all of the individual variants.

A Method for Selectively Recovering Capsid Sequences that haveTransduced Specific Target Cell Populations within Complex TissueSamples.

The AAV genome has two genes—rep, which encodes 4 nonstructural proteinsrelevant for replication (rep78, rep68, rep52 and rep40) and cap, whichencodes three proteins (VP1, VP2, and VP3) that form the shell, orcapsid, of the virus (FIG. 2D). In addition, the cap gene also encodesan accessory protein Assembly-activating protein (AAP) that is requiredfor capsid assembly. Capsid directed evolution methods make use ofreplication competent AAV so that the capsid gene is present in cis(that is, within the viral genome). However, successful production of anAAV capsid variant library depends only upon each variant cap gene beingpackaged by the particular capsid proteins it encodes. Therefore, whileit is useful that the cap gene is present in cis, it is not essentialthat the nonstructural replication (rep) genes be present in cis. Withthis in mind, a replication-incompetent, rAAV genome expressing the capgene and only those regions of the rep gene necessary for expression andsplicing of the capsid gene (FIG. 2D) has been developed. In place ofthe remaining rep sequences, several recombinant elements that provide ameans to selectively recover only those capsid sequences that havefunctionally transduced the target cell population of interest withoutthe need for target cell isolation have been incorporated (FIG. 3A). Toensure that the proteins encoded by the cap are properly expressed, thesplicing donor and acceptor sequences (and all intervening sequences)present within the AAV2 rep gene upstream of the AAV cap gene within therecombinant genome (FIG. 2E) were incorporated. A p41 promoter fragmentfrom AAV5 was used to drive translation from the capsid gene (SEQ ID NO:4, FIG. 15, depicting the entire plasmid: the plasmid backbone, AAVITRs, UBC-mCherry-syn-pA, AAV5 p41 promoter-AAV2 rep splicing seq, AAV9cap, lox71-SV40 polyA-lox66-ITR). To provide rep and AAP helperfunction, an AAV rep/cap helper plasmid was modified by inserting atotal of 5 stop codons within the cap gene within the VP1, 2 and 3reading frame (1 stop codon disrupts VP3, 3 disrupt VP2 and all 5disrupt VP1—FIG. 2F, SEQ ID NO: 5, FIG. 16). These stop codons weredesigned such that they did not disrupt the coding sequence of the AAPprotein, which is encoded within an alternative reading frame.

Selective Recovery of AAV Capsid Sequences from Specific Cre+ CellPopulations.

To facilitate selective recovery of only those capsid sequences thatencode the capsid protein that mediate transduction of a specific targetcell population, a system was designed to take advantage of the largenumber of Cre transgenic mice, rats or other Cre transgenic organismsthat have been (and can be) developed. These lines express Cre under thecontrol of cell-specific promoters such that Cre is present only in asubpopulation of cells within a given organ. This approach incorporatesa Cre recombinase-dependent “switch” that provides a means toselectively recover capsid sequences that have transduced Cre+ targetcells. Cre recombination results in an inversion or deletion (dependingon the configuration the lox sites used—see FIGS. 3 and 4) of a sequencewithin the AAV genome, and a PCR-based recovery strategy was used thatonly amplifies the cap gene sequence from rAAV genomes that haveundergone a Cre-mediated inversion event. This strategy can also beadapted to select for AAV capsids that target cells that had previouslybeen made to express Cre through non transgenic means (e.g., priortransduction with a Cre expressing virus), which could be useful forselection in larger species where Cre transgenes are not available.

An advantage of some of these embodiments is that thisrecombinase-dependent strategy only recovers those sequences that havetransduced the target cell. AAV is a single stranded DNA virus, and itsgenome must be converted to double-stranded DNA (dsDNA) for functionaltransduction. Since Cre only recombines dsDNA, only those capsidsequences that have trafficked properly and have been converted to dsDNAwill be recovered. This increases the selective pressure applied, whichwe anticipate will reduce the number of selection rounds that arenecessary to develop viruses with improved properties.

This application is not limited to using Cre-lox as a recombinase/targetsite system. Other embodiments can include recombinases/integrasesincluding, for example, Flp, phiC31 or Bxb 1. The method can also beadapted for use with recombination-dependent, non-PCR-based recoverystrategies. Furthermore, a recombinant AAV genome lacking most of therep sequences was used to provide space for the lox switch and areporter cassette, a cre-dependent switch could alternatively beinserted within a “replication competent” AAV genome in such a mannerthat it did not disrupt virus gene expression and packaging.

Recent efforts to use rAAV as a vehicle for gene therapy hold promisefor its applicability as a treatment for human diseases based on geneticdefects. rAAV vectors provide long-term expression of introduced genesfrom an episomal genome, although integration of the rAAV genome intothe host chromosomes has been noted (Kaeppel 2013). An additionaladvantage of rAAV is its ability to perform this function in bothdividing and non-dividing cell types including hepatocytes, neurons andskeletal myocytes. rAAV has been used successfully as a gene therapyvehicle to enable expression of erythropoietin in skeletal muscle ofmice (Kessler et al., 1996), tyrosine hydroxylase and aromatic aminoacid decarboxylase in the CNS in monkey models of Parkinson disease(Kaplitt et al., 1994) and Factor IX in skeletal muscle and liver inanimal models of hemophilia. At the clinical level, the rAAV vector hasbeen used in human clinical trials to deliver the cftr gene to cysticfibrosis patients, the Factor IX gene to hemophilia patients (Flotte, etal., 1998, Wagner et al, 1998).

Recombinant AAV is produced in vitro by introduction of gene constructsinto cells known as producer cells. Some systems for production of rAAVemploy three fundamental elements: 1) a gene cassette containing thegene of interest, 2) a gene cassette containing AAV rep and cap genesand 3) a source of “helper” virus genes.

The first gene cassette is constructed with the gene of interest flankedby inverted terminal repeats (ITRs) from AAV. ITRs function to directthe packaging of the gene of interest into the AAV virion. The secondgene cassette contains rep and cap, AAV genes encoding proteins neededfor replication and packaging of rAAV. The rep gene encodes fourproteins (Rep 78, 68, 52 and 40) required for DNA replication. The capgenes encode three structural proteins (VP1, VP2, and VP3) that make upthe virus capsid.

The third element is relevant because AAV-2 does not replicate on itsown. Helper functions are protein products from helper DNA viruses thatcreate a cellular environment conducive to efficient replication andpackaging of rAAV. Adenovirus (Ad) has been used almost exclusively toprovide helper functions for rAAV. The gene products provided by Ad areencoded by the genes E1a, E1b, E2a, E4orf6, and Va.

Production of rAAV vectors for gene therapy can be carried out in vitro,using suitable producer cell lines such as 293 and HeLa. One strategyfor delivering all of the required elements for rAAV production utilizestwo plasmids and a helper virus. This method relies on transfection ofthe producer cells with plasmids containing gene cassettes encoding thenecessary gene products, as well as infection of the cells with Ad toprovide the helper functions. This system employs plasmids with twodifferent gene cassettes. The first is a proviral plasmid encoding therecombinant DNA to be packaged as rAAV. The second is a plasmid encodingthe rep and cap genes. To introduce these various elements into thecells, the cells are infected with Ad as well as transfected with thetwo plasmids. Alternatively, in more recent protocols, the Ad infectionstep can be replaced by transfection with an adenovirus “helper plasmid”containing the VA, E2A and E4 genes. As provided herein, the rep and caparrangements can be in trans for the screening aspects.

While Ad has been used conventionally as the helper virus for rAAVproduction, it is known that other DNA viruses, such as Herpes simplexvirus type 1 (HSV-1) can be used as well. The minimal set of HSV-1 genesrequired for AAV-2 replication and packaging has been identified, andincludes the early genes UL5, UL8, UL52 and UL29. These genes encodecomponents of the HSV-1 core replication machinery, i.e., the helicase,primase, primase accessory proteins, and the single-stranded DNA bindingprotein. This rAAV helper property of HSV-1 has been utilized in thedesign and construction of a recombinant Herpes virus vector capable ofproviding helper virus gene products needed for rAAV production.

The following examples are presented as exemplary embodiments only, andare not intended to be limiting on the scope of the claims. In addition,there are further sections of various embodiments provided between someof the various examples below, as appropriate, and as indicated by thetext and spacing of the document.

Example 1

The Split Rep-AAP and rAAV-Cap-in-Cis Constructs Generate High TiterrAAV.

To test whether the split rep-AAP helper and rAAV-cap-in-cis systemgenerates rAAV virus, a triple transfection of 293 T (ATCC) cells wasperformed with the rep-AAP helper, rAAV mCherry-cap-lox71/66 genome andthe adenoviral helper construct pHelper. Plasmids were transfected at aratio of 2:1:4 (0.263 ug total DNA/cm2 of plated cell surface area),respectively using linear polyethylenimine (PEI) as the transfectionreagent with a N:P ratio of 25. With these constructs, one was able togenerate recombinant virus with an efficiency that was equivalent tothat observed with the standard AAV2/9 rep/cap helper, a rAAV2 genomeexpressing mCherry only and pHelper (FIG. 5A). In contrast, when therep-AAP helper and pHelper were used together with an rAAV genomeencoding mCherry, but not an AAV cap, little to no virus was generated.This confirms that capsid expression in cis was required for rAAVproduction.

Generating the Capsid Libraries: Introducing Short Randomized Sequencesinto Surface Loops of AAV9.

Several strategies can be used to introduce sequence diversity into thecap gene. Examples include, but are not limited to (i) error prone PCR,which introduces mutations at a controllable rate throughout a region ofthe cap gene amplified by PCR, (ii) capsid domain shuffling, wherelibraries are generated through recombination events between fragmentedcapsid sequences generated from a panel of different capsid serotypesand (iii) targeted sequence modification at specific sites using primerswith mixed bases, which generates stretches of randomized sequences atspecific sites within the capsid. Each of these methods has advantagesand disadvantages. In some embodiments, one can use targeted sequencemodification strategy to replace or insert random sequences of sevenamino acids (21 nucleotides) into two different surface loops.

Example 2

To generate libraries of AAV capsid variants, seven amino acids ofrandomized sequence was introduced into the AAV9 capsid. In one library,(452-8r) AA452-8, (VP1 counting) was replaced by randomized sequence. Ina second library, (588i) seven AA of randomized sequence was insertedafter AA588 in the AAV9 capsid. Using this targeted randomizationstrategy the sites can be randomized together in the same library orrandomized sequentially after selection at an individual site.

The library fragments were generated by PCR. AA452-458 of AAV9 werereplaced with 7 random amino acids through the use of a primercontaining a stretch of 21 hand-mixed bases (7×NNK, Primer 1287). Primer1312 was used as a reverse primer. For the 588i library, a stretch of7AA was inserted after AA588 using a primer containing a stretch of 21hand-mixed bases (7×MNN, primer 1286). Primer 1331 was used as a forwardprimer. The PCR conditions reactions were performed using 200 nM of eachprimer, 0.1-0.5 ng of template DNA (pCRII-9R-X/A EK plasmid, SEQ ID NO:6, FIG. 17), 200 um dNTPs, 0.5 ul Q5 Hot Start, High-Fidelity DNAPolymerase (NEB), 10 ul 5× buffer and 10 ul GC enhancer provided by themanufacturer. The template plasmid contained a fragment of the AAV9capsid gene that has been modified to have two unique restriction sites(XbaI and AgeI) flanking the region that was varied (this region createsan overlap with the rAAV9R-X/A-cap-in-cis acceptor plasmid cut with thesame enzymes, see FIG. 5C). In addition, the PCR template fragment wasfurther modified to eliminate a naturally occurring EarI restrictionsite within the capsid gene fragment and insert a KpnI site. Themodification to remove the EarI restriction site provides a way toeliminate any “wild-type” AAV capsid vector sequence contamination fromthe libraries that might arise during cloning by digesting the librarieswith the EarI enzyme. The EarI digestion step may not be necessary ifcare is taken to eliminate the possibility of wt AAV capsid sequencecarry over/amplification. The insertion of the XbaI site caused a K449Rmutation, but the other mutations introduced into the AAV9 sequence aresilent.

To facilitate cloning of the PCR fragments comprising the capsid librarysequences into a recombinant AAV genome, the rAAV-cap-in-cis plasmid wasmodified to introduce the same two unique restriction sites, XbaI andAgeI, within the capsid sequence flanking the region that will bereplaced by the PCR-based libraries (FIG. 5C). In addition, the codingregion between the XbaI and AgeI sites was eliminated to prevent “wt”AAV9R X/A capsid protein production from any undigested vector duringlibrary virus production (AAV9R-delta-X/A-cap-in-cis, SEQ ID NO: 7, FIG.18).

To assemble the PCR library products into the acceptor vector, the PCRproducts can be digested with XbaI and AgeI restriction enzymes and thenligated into the cap-in-cis acceptor construct cut with the sameenzymes. Alternatively, the PCR products and the rAAV-cap-in-cisacceptor vector can be assembled using the Gibson Assembly method(Gibson et al., 2009). Enzymatic assembly of DNA molecules up to severalhundred kilobases. Nature Methods, 6(5), 343-345.doi:10.1038/nmeth.1318). In the examples presented here, the GibsonAssembly method was used to consistently assemble over 100 ng of PlasmidSafe DNase-resistant circular DNA from an assembly reaction made from400 ng of XbaI and AgeI digested, alkaline phosphatase treatedAAV9R-delta-X/A-cap-in-cis vector and 67 ng of library PCR product with30 ul of 2× Gibson Assembly Master Mix (NEB) in a total volume of 60 ul.The reactions were run at 50 C for 120 minutes.

Following Gibson assembly, reaction products were digested with aPlasmid Safe (PS) DNase as directed (Epicenter), which digestslinearized but not circularized DNA molecules. The assembly reactionswere incubated with 1 ul (10U) of PS DNase in a reaction containing 2 ulATP and 7 ul of the reaction buffer supplied by the manufacturer(Epicentre) at 37 C for 20 minutes followed by a heat inactivation stepat 70 C for 30 minutes. This reaction typically yielded over 100 ng ofassembled plasmid (as defined by the measured amount of productremaining after the PS DNase reaction (measured by Qubit dsDNA BroadRange kit from Invitrogen). 100 ng is sufficient to transfect 10 150 mmdishes at 10 ng/dish. It was useful to transfect this amount of therAAV-cap-in-cis library plasmid to minimize number of packaging cellsthat were transfected with multiple copies of the rAAV-cap-in-cisplasmid, which could cause the generation of mosaic capsids. Mosaiccapsids (those having a capsid shell composed of more than one capsidprotein variant) would only carry one capsid variant genome. Therefore,not all of the amino acids within the capsids would be encoded by thecapsid gene within the packaged genome By directly transfecting theassembled DNA, rather than first transforming it into competent cellsand amplifying it in bacteria, it was possible to transfect thepackaging cells with a maximally diverse library (theoretically >1e10unique sequences).

Transfection of 293 Cells for Capsid Library Virus Production.

7-10 150 mm dishes of near confluent 293T cells that had been seeded16-30 hour prior to transfection were typically transfected. In additionto the 10 ng of rAAV-cap-in-cis library vector, 5.7 ug of puc18, 11.4 ugof Rep-AAP helper and 22.82 ug of pHelper (per dish) were co-transfectedusing PEI at a N:P ratio of 25 (see Grieger et al 2006). Thetransfection mix was made in phosphate buffered saline (PBS) and wasincubated at room temperature for 10 minutes and then added drop wiseinto the media. 12-18 hours after transfection, the media on thetransfected cells was exchanged for fresh DMEM supplemented with 5% FBS,1× Pen/Strep and 1× non-essential amino acid mix (Invitrogen). Thismedia was then collected 48 hours after transfection, and replaced withfresh media. At 60 hours post transfection the media and cells werecollected. Virus present in the media was concentrated by precipitationby adding poly(ethylene glycol) and sodium chloride to 8% and 0.5M,respectively. The cell pellets were resuspended in 10 mM Tris, 2 mMMgCl₂ and the viruses were released from the cells by 3 freeze-thawcycles (alternating between a bath made from 100% ethanol and dry iceand a 37 C water bath. After the final thaw at 37 C, the lysates weretreated with 50U of Benzonase for 1 hour at 37 C. The virus precipitatedfrom the media was then collected by centrifugation at 4000×g for 30minutes at 4 C. The pelleted virus from the media was resuspended in thesame Tris-MgCl₂ buffer as above and then combined with the cell lysateviral stock. At this time, deoxycholine (DOC) was added to 0.5% and thevirus stock was incubated at 37 C for an additional 30 minutes. Thevirus stock was then adjusted to 500 mM NaCl and incubated for a further30 minutes before the lysate was cleared by spinning at 4000×g for 15minutes at 25 C. After spinning, the cleared viral stock lysates werepurified over iodixanol (Optiprep, Sigma) step gradients (15%, 25%, 40%and 60% as described by Ayuso et al 2010). Viruses were then sterilefiltered and dialyzed with Amicon Ultra 100K Centrifugal filters asdirected (Invitrogen) and concentrated in PBS. Virus titers weredetermined by measuring the number of DNasel-resistant genome copies(GCs) using qPCR and a linearized plasmid as a control (Gray et al2011).

The virus production was halted at 60 hours post-transfection to reducethe likelihood of secondary transduction of the producer cells by therAAV-cap-in-cis virus that is released into the medium.

Secondary transduction of cells that were successfully transfected withall of the plasmids necessary for virus production may lead to thegeneration of viruses from more than one capsid sequence (i.e.,mosaics). Effort can be taken to minimize mosaic virus production toensure that each capsid gene is packaged only into the physical capsidvariant that it encodes.

Discussion of Additional Embodiments and Further Examples

Based on the results from the initial examples presented below, it isexpected that this system can be used in conjunction with any transgenicline expressing a recombinase in the target cell type of interest todevelop AAV capsids that more efficiently transduce that target cellpopulation. Applications include, but are not limited to, developingcapsids that are more efficient at transducing specific cell types inany organ after IV AAV administration, targeting specific populations ofneurons after intraparenchymal brain injections, improving neuronaltransport (anterograde or retrograde), targeting tumor cells,hematopoetic stem cells, insulin producing beta cells, lung epithelium,skeletal or cardiac muscle. Thus, the selection methods provided hereincan be applied for one or more these aspects.

In addition, the approach can be used to select for viruses that targethuman cells in human/mouse chimeric animals (the human cells would bemade to express Cre prior to in vivo delivery). This last example can beuseful in the successful development of efficient vectors for genetherapy applications as there is evidence that the AAV serotypes thatfunction best in animal models may not always function with the sameefficiencies in humans (Lisowski et al 2013). Therefore, it can beadvantageous to select for viruses that most efficiently transduce thetarget human cell population in the in vivo context of an animal model.

In addition, the method can be used in conjunction with any virusdelivery method (e.g., intravenously, SC, IP, intramuscular, intranasal,i.c.v, intrathecal, oral or intracranial/intraparenchymal braininjection). In some embodiments, the vector can be delivered via anyroute including, but not limited to: oral, intravenous, intraarticular,intracardiac, intramuscular, intradermal, topical, intranasal,intraparitoneal, rectal, sublingual, subcutaneous, epidural,intracerebral, intracerebroventricular, intrathecal, intravitreal orsubretinal administrations.

Although the examples herein have used AAV9 as a starting point, anynaturally occurring or previously engineered AAV capsid could also beused as a starting point for selection using this approach. Furthermore,this method could also be useful for identifying other coding ornon-coding sequences within an AAV or other viral genome that influencedtransduction of recombinase expressing cells. Preferred examples includeselecting for sequences within the AAV genome that increase conversionof the viral genome to dsDNA, increase the persistence of viral genomesby facilitating recombination or circularization, increase theefficiency of integration of the viral genome into a favored site(s) inthe cellular genome or sequences that influence gene expression in thetarget cell population.

In some embodiments, provided herein is the use of CREATE (CreRecombinase-based AAV Targeted Evolution), a novel platform for theselective recovery of capsid sequences that transduce Cre⁺ target cellpopulations. Using CREATE, it was possible to develop several new AAVcapsid variants with useful properties, including one, AAV-PHP.R2, thatmediates efficient retrograde transduction within the brain as early asseven days post administration, and a second variant, AAV-PHP.B thatcrosses the adult mouse blood brain barrier (BBB) and transduces avariety of CNS neural cell types with an efficiency that is at least40-fold greater than AAV9, the current standard for systemic delivery.In addition, whole animal tissue clearing using PARS-based CLARITY (Yanget al., 2014b) as a more rapid method for assessing serotype tropism atthe cellular level and as a method to study individual cell morphologyin the brain when combined with low-dose systemic AAV-PHP.B delivery isprovided. Used together, transduction mapping in intact tissues and theCre-based capsid selection method presented provide a novel platformthat should facilitate further custom virus development.

Example 3

In Vivo Selection Using GFAP-Cre Transgenic Animals

AAV capsids were developed that more efficiently transduce cells withinthe CNS of adult mice after IV injection. For this purpose, transgenicmGFAP-Cre mice were used that express Cre specifically within astrocytesand neural stem cells (NSCs) in the adult brain and spinal cord. Thecapsid libraries (1.2e11 GC) were delivered intravenously (IV) to micethrough the retro-orbital sinus. 7-8 days later, the DNA was collectedfrom the entire brain and spinal cord and recovered rAAV capsidsequences from GFAP-Cre+ cells (FIG. 6). Vector DNA was recovered fromone hemisphere of the brain and half of the spinal cord using 4 ml ofTrizol (Invitrogen). The manufacture's protocol was followed, and theaqueous, RNA-containing fraction was precipitated with isopropanol andsubjected to three washes in 70% ethanol made with water (all water usedfor PCR recovery of capsid sequences in this protocol is treated with UVusing a UV light box for 10-15 minutes prior to use). The precipitatedmaterial was then resuspended in 10 mM Tris pH8.0. In addition to RNA,this fraction also contains a significant fraction of the viral genomeas well as some mitochondrial DNA. To eliminate the RNA, which reducedthe efficiency of the PCR-based recovery of capsid sequences, thesamples were treated with 1 ul of RNase (Qiagen) overnight. Alternativestrategies for selective recovery of viral genomes away from theanimal's genomic DNA could also be used, e.g., the HIRT extractionprotocol (Hirt 1967), sized-based gel-purification, sequences specificcapture/hybridization methods or selective digestion of the mousegenomic DNA by PS DNase following digestion with a restriction enzymethat does not cut the rAAV-cap-in-cis genome.

Capsid sequence recovery was performed in a Cre-dependent manner usingprimers 1253+1316. PCR conditions were 20-28 cycles with 95 C 20 sec/60C for 20 sec/72 C for 30 sec using Q5 Hot Start High-fidelity DNAPolymerase. The PCR product was then diluted 1:10 and then used as atemplate for a second, PCR reaction that generated the X to A fragment(using primers 1331+1312) that was cloned back into therAAV9R-deltaX/A-cap-in-cis acceptor construct to generate the next roundof library virus using the same methods describe above. 1 ul of theGibson Assembly reactions was then diluted 1:10 and transformed intoSure2 competent cells (Agilent) as directed by the manufacturer. Atleast 10 colonies/library were picked 12-16 hours later, DNA wasisolated by miniprep kit (Qiagen) and the clones were sequenced.Alternatively, DNA from the clones can be amplified by PCR using primers1253 and 1312 and sequenced directly, eliminating the need to performmini plasmid DNA preps.

After the first round of selection all of the clones sequenced for bothlibraries were unique. Therefore, a second selection round was performedto further enrich for the most potent sequences. The assembledrAAV-cap-in-cis library regenerated after the first round of selectionwas used to generate a second round of virus which was then injectedinto a second batch of GFAP-Cre+ mice as described above. After thesecond round, two sequences, G2B13 and G2B26 showed evidence ofenrichment (Table 2).

TABLE 2 Enriched Sequences from GFAP-Cre in vivo selection % of MouseSelection total Variant line Delivery rounds site 7mer DNA sequence(s)7mer AA sequence(s) clones G2B-13 GFAP- IV 2 452-8 CAGTCGTCGCAGACGCCTAGGQSSQTPR 18% Cre (SEQ ID NO: 48) (SEQ ID NO: 54) G2B-26 GFAP- IV 2 588ACTTTGGCGGTGCCTTTTAAG TLAVPFK 27% Cre (SEQ ID NO: 49) (SEQ ID NO: 1)TH1.1- TH-Cre intra- 1 + 1 452-8 + ATTCTGGGGACTGGTACTTCG ILGTGTS (452-8)18% 32 crainial 588 (SEQ ID NO: 50) (SEQ ID NO: 55) (striatum)ACGCGGACTAATCCTGAGGCT TRTNPEA (588)  9% (SEQ ID NO: 51) (SEQ ID NO: 56)TH1.1- TH-Cre intra- 1 + 1 452-8 + ATTCTGGGGACTGGTACTTCG ILGTGTS (452-8)18% 35 crainial 588 (SEQ ID NO: 52) (SEQ ID NO: 57) (striatum)AATGGGGGGACTAGTAGTTCT NGGTSSS (588) 36% (SEQ ID NO: 53) (SEQ ID NO: 58)

To test the variants recovered, the sequences were cut with BsiWI andAgeI and ligated into an AAV2/9R-X/A rep/cap helper (AAV2/9 rep/caphelper modified with the AAV9R-X/A capsid sequence from rAAV-cap-in-cisplasmid) also cut with BsiWI and AgeI and transformed into DH5alphacompetent cells (NEB). Plasmid DNA was purified using an EndofreePlasmid Maxi Kit (Qiagen). The resulting rep/cap plasmids carrying thenovel variant sequences, or AAV2/9 rep/cap as a control, were then usedto package a rAAV genome containing a dual eGFP-2A-luciferase reportercassette driven by a ubiquitous CAG promoter(rAAV-CAG-eGFP-2A-Luc-WPRE-SV40 pA). The novel capsids packaged thegenome with efficiencies comparable with AAV9 (FIG. 7). 1e12 GC of eachvector was injected IV into individual adult female C57B1/6 mice. Sixdays later, the mice were perfused with 4% paraformaldehyde in 100 mMphosphate buffer and the brains were examined for eGFP fluorescence.

Remarkably, transduction by the G2B26 variant was efficient enough thatthe native eGFP fluorescence throughout the intact brain could be seenwith a 1× objective on an epifluorescence microscope (FIG. 8A). At thissame exposure setting, little to no eGFP fluorescence is evident in thebrain from the mouse injected with AAV9. In sections prepared from abrain from a mouse injected with G2B26, transduction of neurons and gliain all regions examined in the brain and spinal cord were seen (FIGS. 8and 9). In certain thalamic nuclei, over 90% of the NeuN+ cell bodiesexpressed GFP (FIG. 9G). Transduction of motor neurons in the ventralspinal cord was also robust (FIG. 9F). Numerous Sox2+ glia expressed GFP(FIG. 9I). The G2B13 variant also transduced astrocytes and neurons moreefficiently than AAV9, but the effect was not as dramatic as compared tothe transduction by G2B26 (FIGS. 8 and 9A-C). The G2B13 variant showedstrong transduction of fiber tracts in the dorsal brain stem (FIG. 9B)and spinal cord (FIG. 9C) as well as robust liver transduction (FIG.8C). It also appears that the G2B26 variant provides more rapid onset ofexpression in the CNS than AAV9. Transduction by AAV9 transduction atsix days post-injection was weak. Stronger expression was observed percell and more eGFP expressing cells with the same dose of AAV9 at 21days post injection.

Since rapid unpackaging has been proposed to be an important componentof transduction efficiency and viral genome persistence (Wang et al.2007), recovering capsid sequences soon after injection (in this case7-8 days) may be an important component of a successful selection.

In the example above, the number of cycles can be determined empiricallywith the optimal number of cycles being within a range that yields moreproduct from samples taken from Cre+ cells/animals than from sampleslacking Cre+ cells. If the PCR reaction is allowed to continue past thisoptimal range by performing too many cycles, products may be recoveredeven from Cre negative samples. It can be desirable to avoid doing toomany cycles.

Example 4 In Vivo Selection for Improved Retrograde Transduction UsingTH-Cre Animals

In a second test of the Cre-dependent selection platform, it was askedwhether one could generate novel AAV serotypes that lead to moreefficient transduction of TH+ neurons in the substantia nigra (SN)compact part after virus injection into the striatum, a structure thatreceives axons from TH neurons. This selection scheme is designed todevelop AAVs that are capable of rapid retrograde transport andtransduction of TH+ and non-TH+ cells.

The same libraries were used as initially generated for the Example 3selection and injected 0.6 ul of the virus bilaterally into the striataof adult TH-Cre+ male mice using the stereotaxic coordinates 0.7 mmrostral, 2.0 mm lateral and 3.0 mm ventral from Bregma. 10 days later,the region containing the SN was collected and isolated virus DNA fromthe tissue as described above. For these dissections, the mCherryreporter expressed from the rAAV-cap-in-cis genome aided in theidentification of the SN (FIG. 10C) and the confirmation that the viruslibraries injections had targeted the desired areas (FIG. 10B). VirusDNA was obtained from the SN-containing tissue sample using Trizol(Invitrogen) as described above and the same Cre-dependent PCR strategywas used to selectively recover those capsid sequences that led to thetransduction of TH+ neurons (FIG. 10D). Using primers that amplifycapsid sequences from all genomes regardless of recombination status(1253+1267) demonstrate that the viral sequences were also present inthe Cre-controls (FIG. 10D, lower panel). Sequences recovered throughthe Cre-recombination dependent strategy were cloned back into therAAV-cap-in-cis library acceptor as described above in Example 3.

Colonies were picked for sequencing. After the first round of selection,all of the tested sequences were unique, so a second round of selectionwas performed as described above. In addition to continuing with thelibraries modified at the two individual sites, combinatorial librarieswere also made by mixing all of the sequences recovered at the 452-8replacement site with the sequences recovered at the 588 insertion siteusing the PCR strategy outlined in FIG. 10. Capsid virus libraries fromthe recovered sequences were prepared, selected again in TH-Cre mice andrecovered as described above.

After the second selection round in the combinatorial library, severalsequences at both randomization sites showed evidence of enrichment.Several novel capsid sequences were selected to test as individualvariants. The sequences were cloned into an AAV2/9R-X/A rep/cap helperusing unique BsiWI and AgeI sites present in both vectors. The resultingrep/cap plasmids carrying the novel variant sequences, or AAV2/9 rep/capas a control, were then used to package a single stranded (ss)rAAV-CAG-GFP-W-pA genome. The novel capsids packaged the genome withefficiencies comparable with AAV9 (FIG. 7). 7e9 VGs (in 0.5 ul) of eachvirus was injected individually, and bilaterally, into adult C57B1/6mice using the same stereotaxic coordinates described above. 7 dayslater, mice were given an overdose of Euthasol and killed by cardiacperfusion with 4% PFA as described above. At this time, there were fewif any GFP+/TH+ neurons in the SNc of mice that received an injection ofthe control virus, AAV9:CAG-GFP-W-SV40 pA. In contrast, there werenumerous GFP+/TH+ neurons present in the mice given injections of thesame rAAV genome packaged into the novel clones TH1.1-32 (FIG. 12D) orTH1.1-35 (FIG. 23D)

A listing of the primers used in examples 3 and 4 is provided in Table 1below:

TABLE 1 Primer Purpose Sequence 1253 Cre-dependentCAGGTCTTCACGGACTCAGACTATCAG amplification, forward (SEQ ID NO: 16) 12549R-X/A delta, reverse CAACCGGTAATAGTTCTAGAGAGATAGTACAAGTATTGGTCGATGAGTG(SEQ ID NO: 37) 1255 9R-X/A delta, forwardCTCTCTAGAACTATTACCGGTTGGGTTCAAAACCAAGGAATACTTC (SEQ ID NO: 38) 1267Library recovery, non- GTCCAAACTCATCAATGTATCTTATCATGTCTGrecombined, reverse (SEQ ID NO: 39) 1280 VP1 stop, reverseGAGTCAATCTGGAAGTTAACCATCGGCA (SEQ ID NO: 40) 1281 VP1 stop, forwardGATGGTTAACTTCCAGATTGACTCG (SEQ ID NO: 41) 1283 VP2 stop, reverseGACTACTCTACAGGCCTCTTCTATCCAG (SEQ ID NO: 42) 1284 VP2 stop, forwardGATAGAAGAGGCCTGTAGAGTAGTCTCC (SEQ ID NO: 43) 1285 VP3 stop, reverseCATCGGCACCTTAGTTATTGTCTG (SEQ ID NO: 44) 1286 VP3 stop, forwardGACAATAACTAAGGTGCCGATGGAGTGG (SEQ ID NO: 45) 1286 Site 588GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNMNN randomization, reverseMNNMNNMNNMNN (SEQ ID NO: 23) 1287 Site 452-8CATCGACCAATACTTGTACTATCTCTCTAGAACTATTNKKNKKNKKNKK randomization, reverseNKKNKKNKKCAAACGCTAAAATTCAGTGTGGCCGGA (SEQ ID NO: 22) 1312Site 452-8, reverse GGAAGTATTCCTTGGTTTTGAACCCA and X/A fragment(SEQ ID NO: 19) generation, reverse 1316 Library recovery, Cre-CAAGTAAAACCTCTACAAATGTGGTAAAATCG dependent, forward (SEQ ID NO: 17)(reversed by recombination) 1331 Site 588, forward andACTCATCGACCAATACTTGTACTATCTCTCTAGAAC X/A fragment (SEQ ID NO: 18)generation, forward 1352 Combinatorial libraryGTCTCTGCCGGTACCTTGTTTGCCAAAAATTAAAGATCCA generation, Earl to(SEQ ID NO: 46) KpnI mutation insertion, Rev 1353 Combinatorial libraryGCAAACAAGGTACCGGCAGAGACAACGTGGATGCGGACA generation, Earl to(SEQ ID NO: 47) KpnI mutation insertion, For

By using the platform for selection provided herein, it was possible todeveloped several capsids that provide enhanced, widespread geneexpression in the CNS.

Notably, one capsid (AAV-PHP.R2) was capable of rapid, retrogradetransport within CNS neurons after intracerebral injection, whileanother capsid (AAV-PHP.B) transduced cells throughout central nervoussystems with 40-90-fold greater efficiency than AAV9 when deliveredsystemically. AAV-PHP.B transduces both neurons and glia and istherefore well suited for gene transfer to global CNS neural cell typesincluding neurons, astrocytes and oligodendrocytes.

Given the large collection of cell type-specific Cre transgenic lines,the present capsid-selection platform is a valuable resource forcustomizing gene delivery vectors for biomedical applications.

Example 5

Within the rAAV cap-in-cis recombinant genome, two elements wereintroduced to facilitate the selection. The first is an mCherry reportercassette, having a 398 base pair promoter fragment from the ubiquitin Cgene (UBC), the 711 bp mCherry cDNA, and a 118 bp 3′ untranslated regioncontaining a 51 bp synthetic poly adenylation (polyA) sequence (Levittet al., 1989). The second, and more relevant element is a Cre-dependent“switch”, having a pair of inverted, modified loxP sites (lox71 andlox66) (Araki et al., 1997) flanking a SV40 polyA sequence downstream ofthe cap gene. This foxed element created a Cre-invertible sequence thatallows for the selective PCR amplification and recovery of only thosecap sequences contained within the AAV genomes that have transduced Cre⁺cells (FIG. 22A).

To provide rep, but not cap, gene function in trans, an AAV2/9 REP-CAPhelper plasmid was modified by inserting five in frame stop codonswithin the reading frame for the capsid proteins, VP1, VP2 and VP3.These stop codons were designed to disrupt capsid protein expression,but not alter the amino acid sequence of the assembly activating protein(AAP), which is expressed from an alternative reading frame within thecap gene (FIG. 22B) (Sonntag et al., 2010). In this way, the modifiedREP-AAP helper plasmid continues to provide all of the AAV gene productsin trans, save for capsid protein expression. To test whether this splitrAAV-CAP-in-cis-lox and REP-AAP helper system efficiently generatesrAAV, a triple transfection of HEK 293T cells was performed with therAAV-CAP-in-cis genome, the REP-AAP helper, and the adenoviral helperplasmid, pHelper. Importantly, with these plasmids, it was possible togenerate recombinant virus with an efficiency that was equivalent, ifnot greater than, that observed when an AAV2/9 REP-CAP helper was usedto package a rAAV genome encoding mCherry (AAV-UBC-mCherry) (FIG. 22C).

In contrast, when the AAV REP-AAP helper was used to packageAAV-UBC-mCherry, lacking the cap gene in cis, little to no virus wasgenerated, confirming that capsid protein expression from therAAV-CAP-in-cis-lox vector was required for rAAV production. Usedtogether, the rAAV-CAP-in-cis-lox and AAV REP-AAP helper provided anovel platform, which is here below termed CREATE, for selective capsidsequence recovery from genetically defined populations of cells withincomplex tissue samples.

Example 6

Two AAV9-based capsid libraries were generated by PCR using a mixed baserandomization strategy. One library was made by inserting 7 amino acidsof randomized sequence between AA588-9 (VP1 position) of the AAV9 capsidand another with 7 amino acids of randomized sequence replacing AA452-8of AAV9. The cloning strategy was designed such that the recoverable PCRproduct would contain only the stretch of amino acids spanning thevariable regions (sequences between AA450 and AA592), which encompassesa significant portion of the surface exposed amino acids, while the restof the capsid sequence within the backbone vector remains unmodified.Library fragments were then cloned into the rAAV-delta-cap-in-cis vectorand assembled products were directly transfected into packaging cells toproduce virus, bypassing the primary bottleneck of librarydiversification, bacterial transformation. With this approach, thelibrary diversity is limited by the number of transfected cells, ratherthan the number of bacterial transformants resulting in an estimateddiversity of 1×10⁷-1×10⁸ unique sequences. Using this approach, it waspossible to achieve yields of 5-10×10¹¹ VGs.

Vectors that mediate efficient retrograde transduction of neurons, i.e.,the uptake of vector by axons and transport back to the nucleus, aredesired for neuronal circuit tracing and intersectional approaches forcircuit-specific gene expression, and may also have uses for clinicalgene delivery. While viruses such as recombinant rabies and herpessimplex virus (HSV), exhibit highly efficient retrograde transductionand are useful for short-term circuit tracing studies, their long-termtoxicity precludes their use for longitudinal experiments or experimentswhere their impact on cellular health would cofound (e.g. optogenetics,aging, neurodegeneration studies). For long-lasting gene expression,AAVs capable of efficient retrograde transduction would be highlyvaluable as they would allow the extensive tool-set available in therAAV genome format to be applied to applications requiring retrogradetransduction (NIH Brain Initiative Working Group, 2013).

Example 7 In Vivo Selection for AAV Variants with Enhanced RetrogradeTransduction in the Rodent CNS

Several AAV serotypes have been shown to mediate retrograde transductionof neurons in the CNS with varying efficiencies (Aschauer et al., 2013;Castle et al., 2014a; Castle et al., 2014b; Cearley and Wolfe, 2007;Hutson et al., 2012; Low et al., 2013; Salegio et al., 2013; Samaranchet al., 2012). To develop AAV capsids with improved retrogradetransduction, an in vivo selection for capsids that transduced TH⁺dopaminergic neurons in the substantia nigra via retrograde transportfrom their axons within striatum (Smith and Bolam, 1990) was set up. TheAAV-CAP-in-cis-lox 452-8r and 588i libraries were separately injectedinto the striata of adult TH-Cre⁺ mice. 10 days later the tissuesurrounding the substantia nigra (SN) (FIG. 23A) was dissected andisolated viral DNA. For these dissections, the mCherry reporterexpressed from the AAV-cap-in-cis library vectors aided in theidentification of the SN as the SN pars reticulata (SNr) was easilyidentified from the mCherry⁺ axons that project to the SNr from thestriatum. mCherry expression in the striatum confirmed that the viruslibrary injections had been properly targeted (FIG. 23A). After thefirst round of selection, 10 clones from each library were sequenced andit was found that all of the tested sequences were unique, so a secondround of selection was performed. To further diversify the librariesafter the initial round of enrichment, combinatorial libraries were madeby mixing all of the sequences recovered from the 452-8r library withall of the sequences recovered from the 588i library by PCR (see FIG.27D). Viral capsid libraries from the combinatorial library wereprepared and selected again in TH-Cre mice as described above. After thesecond selection round, several sequences at both randomization sitesshowed evidence of enrichment.

The most highly enriched variant, PHP.R2, was further characterized bytesting it individually (see Table 3 for sequence information andenrichment data).

TABLE 3 Variant Selection Route Rounds Site(s) 7mer DNA sequence AA seq.% PHP.R2 TH i.c. 1 + 1 452-8r ATTCTGGGGACTGGTACTTCGILGTGTS (SEQ ID NO: 55) 18% 588i (SEQ ID NO: 50) NGGTSSS (SEQ ID NO: 58)36% AATGGGGGGACTAGTAGTTCT (SEQ ID NO: 53) PHP.A GFAP i.v. 2 588iTATACTTTGTCGCAGGGTTGG YLSQGW (SEQ ID NO: 60) 40% (SEQ ID NO: 59) PHP.BGFAP i.v. 2 588i ACTTTGGCGGTGCCTTTTAAG TLAVPFK (SEQ ID NO: 1) 27%(SEQ ID NO: 49)

Table 3 lists the AAV-PHP variants, the Cre transgenic line used toperform the library selection, the route of administration and thenumber of selection rounds used to enrich for the improved variants. 1+1refers to one round of selection of the two separate libraries and thenan additional round of selection of the combinatorial library. The sitewithin AAV9 that was modified in each recovered variant is listed as isthe 7mer DNA sequence(s) and amino acid sequence(s) (AA seq.) that aremodified in each capsid variant. The number of occurrences of theenriched sequence as a percentage of the total number of clonessequenced is also given.

AAV-PHP.R2 was used to package a single stranded (ss) AAV-CAG-GFP genomeand injected it into the striatum of adult mice. Notably, after only 7days, robust GFP expression was observed at the striatal injection site(FIG. 23B) as well as at sites distant to the injection that are knownto send projections to the striatum including the SNc (FIGS. 23C and23D), the cortex (FIGS. 23E and 23F), the thalamus (FIG. 23G) and theamygdala (FIG. 23H). These results demonstrate that AAV-PHP.R2 canprovide rapid retrograde transduction of several distributed neuronalpopulations.

Example 8 In Vivo Selection for AAV Variants Capable of Widespread CNSTransduction Following Systemic Administration

The present example examines the development of AAV capsids that moreefficiently transduce cells throughout the CNS. Several AAVs, mostnotably AAV9, rh.10 and rh.8, transduce CNS neurons and glia afterneonatal or adult systemic, intravenous delivery (Duque et al., 2009;Foust et al., 2009; Gray et al., 2011; Samaranch et al., 2011; Yang etal., 2014a). While systemic rAAV administration with these serotypes iscapable of widespread CNS delivery, the transduction efficiency issignificantly reduced compared to that achievable in other organs suchas liver, heart or skeletal muscle (Pulicherla et al., 2011). Thepresent example demonstrates the use of the CREATE platform to developcapsids that more efficiently transduce the CNS globally. This was donegiven the important roles astrocytes play in the pathogenesis ofneurodegenerative disease, together with the baseline tropism of AAV9for astrocytes.

The AA452-8r and AA588i capsid libraries described above were deliveredinto transgenic mGFAP-Cre mice that express Cre from the mouse GFAPpromoter, which is expressed within astrocytes and neural stem cells(NSCs) in the adult brain and spinal cord (Garcia et al., 2004). 1×10¹¹VG of each capsid library were injected into separate adult GFAP-Crepositive mice and GFAP-Cre negative mice as controls. Seven days later,virus DNA from the brains and spinal cords and recovered capsidsequences from viral genomes that had undergone Cre-mediatedrecombination by PCR were isolated. The recovered fragments were clonedback into the rAAV-CAP-in-cis-lox acceptor vector, and clones from eachlibrary were picked at random for sequencing. As observed in the firstround of the TH-Cre selection, all of the tested sequences recoveredfrom both libraries after the first round were unique. After the secondround, a single sequence, designate as AAV-PHP.B, was identified fromthe 588i library and showed signs of enrichment.

To assess the AAV-PHP.B variant individually, this capsid or AAV9 wasused to package a dual-GFP and firefly Luciferase reporter vector,ssAAV-CAG-GFP-2A-Luc. AAV-PHP.B packaged the recombinant genome with anefficiency similar to AAV9 (FIGS. 27A-27E). Next, 1×10¹² VG ofssAAV-CAG-GFP-2A-Luc packaged into AAV-PHP.B or AAV9 was delivered intoadult mice by IV injection and assessed transduction by GFP expressionthree weeks later. Remarkably, this variant transduced the entire CNSwith high efficiency as indicated by immunostaining for GFP (FIGS. 24Aand 24C) and analysis of native eGFP fluorescence in several brainregions (FIGS. 24B, 24D, and 24G), the spinal cord (FIGS. 24D and 24G)and retina (FIG. 24E-24F). Native GFP fluorescence remained dramaticallyincreased over AAV9 even when 10-fold less AAV-PHP.B was delivered (FIG.25A-right and 25D). In stark contrast with AAV9, which sparsely labelsneurons and glia, individual transduced astrocytes were difficult todiscern in mice that received 1×10¹² VG AAV-PHP.B, but could be seen inanimals that received 10-fold less virus (FIGS. 24A-24B and FIG. 25A).AAV-PHP.B also transduced cerebellar Purkinje cells with strikingly highefficiency as demonstrated by co-localization of GFP and Calbindinimmunostaining (FIG. 24C). Taking advantage of recent advances inCLARITY-based tissue clearing (Yang et al., 2014b), the native eGFPfluorescence was imaged through several hundred micron thick sections oftissue from the cortex, striatum and ventral spinal cord. These 3Drenderings further demonstrate the efficiency of transduction by theAAV-PHP.B variant and confirm tissue clearing (Chung and Deisseroth,2013; Tomer et al., 2014; Yang et al., 2014b) as a means of assessingthe three-dimensional distribution of transduced cells within the brain(FIG. 24G).

To quantify CNS transduction by this variant as compared to AAV9, thenumber of viral genomes present in several brain regions 25 days postinjection were measured. Brain and spinal cord transduction by AAV-PHP.Bwas between 40 and 92-fold more efficient than AAV9, depending on theregion examined (FIG. 24H), while outside of the CNS, the AAV-PHP.Bvector transduced several peripheral organs less efficiently than AAV9(FIG. 24I). Remarkably, in all regions other than the cerebellum, thenumber of viral genomes detected in the CNS in mice treated withAAV-PHP.B was similar that observed in the liver and greater than thatseen in the other peripheral organs examined. In stark contrast, afterAAV9 transduction, the number of AAV genomes detected within any of theCNS regions examined was at least 120-fold less than the number found inthe liver. Therefore, while the tropism of AAV-PHP.B was not CNSspecific, the enhanced transduction exhibited by this vector was CNSspecific. In an initial selection using CREATE in GFAP-Cre mice anAAV9-based variant, AAV-PHP.A, was identified that exhibited both moreefficient astrocyte transduction as well as reduced tropism for severalperipheral organs (FIG. 28A-28E). Based on these results, AAV-PHP.Bappears applicable for non-invasive, CNS-wide gene transfer in theadult.

AAV9 preferentially transduces astrocytes when delivered systemically toadult animals, but it also transduces neurons in several regions (FIG.24A and FIG. 25A, Foust et al 2009, and Yang et al 2014). To examine thecell types transduced by AAV-PHP.B, the colocalization of GFP expressionwith proteins expressed in specific cell populations was analyzed.AAV-PHP.B transduced GFAP⁺ astrocytes (FIG. 25A), CC1⁺ oligodendrocytes(FIG. 25B), NeuN⁺ neurons (FIG. 25C and FIG. 25D) but not IBA1⁺microglia (FIG. 25E).

Several cell types of clinical importance are also targeted with highefficiency including TH⁺ dopaminergic neurons in the SNc (FIG. 25F),spinal motor neurons (FIG. 34D and FIG. 24H) and striatal medium spinyneurons (FIG. 25D). In addition, several interneuron populations werealso transduced (FIG. 25G-25J), although strong eGFP fluorescence wasrarely found to colocalize with cells with Calretinin staining (FIG.25J).

In sum, adult IV administration of AAV-PHP.B can be used to target, withhigh efficiency, numerous CNS cell types of scientific and clinicalinterest.

Tissue Clearing for Serotype Tropism Characterization and 3D CellPhenotyping

Because CLARITY allows for the 3D imaging of cells in thick sections orintact tissue (Chung et al 2013; Yang et al. 2014) and AAV-PHP.Btransduces numerous glial and neuronal cell types in the brain, whetherCLARITY could be used together with a low dose of AAV-PHP.B to providerandom, Golgi-like labeling of neural cells in the CNS was examined. Toevaluate this approach, 1×10¹⁰ VG of AAV-PHP.B expressing GFP wasdelivered into adult mice by IV injection. These mice were perfusioncleared and native GFP fluorescence in the brains of the mice wasimaged. Individual neurons, astrocytes and endothelial cells werevisible and could be imaged through at least 400 um of cleared tissue(FIG. 26D).

This approach can be useful for studying the morphology of individualcells in normal and diseased states. This approach can be used toco-express a reporter along with any of the following examples ofgenetic elements to investigate the effects of said genetic element oncell morphology or connectivity in vivo: a gene encoding a protein ofinterest; Cre, or another recombinase, for conditional gene modificationin transgenic animals harboring a foxed target allele(s); conditional,foxed, alleles to transgenic animals made to express Cre in a definedtarget cell population; a gene knockdown cassettes containing a suitablepromoter and shRNA or miRNA, or an endogenous miRNA sponge or decoy.Given the ease of adjusting the labeling/gene modification frequency bymodulating the amount of virus administered, this vector could also beused to address questions related to cell autonomy by generating geneticmosaics.

Whole animal tissue clearing using PARS-based CLARITY may also be usefulfor the assessment of AAV tropism at a cellular level. This is typicallya labor-intensive process that requires processing, mounting and imagingindividual thin (1-100 micron) sections of tissue from each organ. Thepotential whole animal tissue clearing to reduce this burden wasexplored. 1×10¹² VG of ssAAV-CAG-GFP-2A-Luc packaged into AAV-PHP.B orAAV9 was delivered into adult mice by IV injection. Three weeks later,all of the tissue in the mice was cleared using the PARS-based CLARITYmethod described in Yang et al. (2014) and used confocal imaging and 3Dimage reconstruction (Imaris software, Bitplane) to assess native GFPexpression as a reporter of vector transduction. In several organs,including skeletal muscle, lung, pancreas, and the liver, the mice thatreceived AAV-PHP.B showed a reduction in the expression of GFP ascompared to the mice that received an equivalent dose of AAV9 (FIG. 29).The reduced GFP expression in several peripheral organs observed withAAV-PHP.B as compared with AAV9 appears consistent with the number ofVGs detected for each vector in these same peripheral organs (FIG. 24I).Note the GFP positive nerve fibers present in the muscle and pancreas ofmice injected with AAV9 and AAV-PHP.B.

rAAV labeling combined with CLARITY will be a useful approach forstudying the 3D morphologies of peripheral nerves.

The following aspects apply to the experiments outlined in Examples 7and 8 above:

Mice

5-week-old female C57B1/6 mice were purchased from the Jackson Labs(Maine). GFAP-Cre mice expressing Cre under the control of the mouseGFAP promoter (Garcia et al., 2004) and TH-Cre mice (Savitt et al.,2005) were from the Jackson Labs. In vivo selection was performed inadult mice of either sex.

Plasmids

The rAAV-cap-in-cis-lox plasmid contains the following elements clonedinto a vector containing AAV2 ITRs (Balazs et al. 2011). An mCherryexpression cassette (398 bp fragment of the human UBC gene upstream ofthe mCherry reporter followed by a 48 bp synthetic polyAsequence—(Levitt et al., 1989) followed by the AAV9 capsid cassette.Expression of the AAV9 capsid gene was placed under the control of thep41 promoter sequence from AAV5 (1680-1974 of GenBank AF085716.1; Qiu,Nayak Pintel 2002 and Farris and Pintel 2008) and splicing sequencestaken from the rep gene from the AAV9 packaging plasmid (U. Penn). ASV40 polyA sequence flanked by inverted lox71 and lox66 sites was placeddownstream of the AAV9 capsid. The rAAV-cap-in-cis-lox plasmid wasmodified to introduce two unique restriction sites, XbaI and AgeI,within the capsid sequence flanking the region that was replaced by thePCR fragment. The insertion of the XbaI site caused a K449R mutation andthe mutations required to insert the AgeI site were silent.

In the second iteration of the library construction (used in the secondGFAP-Cre capsid selection that yielded PHP.B), two modifications weremade to reduce contamination of the libraries by AAV9 or the startingAAV9R X/A capsid. First, the coding region between the XbaI and AgeIsites was eliminated in the plasmid used for the capsid library cloning(rAAV-Δcap-in-cis acceptor) to eliminate any potential carryover ofundigested plasmid. Second, the PCR fragment covering the capsid libraryvariable region between the XbaI and AgeI sites was modified to remove aunique EarI restriction site (xE) within this region of AAV9 and inserta unique KpnI site. The modified xE fragment was TA cloned into pCRIIIto generate pCRII-9Cap-xE, which served as the template for our laterlibrary PCR fragments. Eliminating the EarI site provided a secondaryprecaution allowing for the digestion of any contaminating AAV9sequences recovered by PCR. It was not necessary to use this digestionstep as taking standard PCR precautions including UV treating reagentsand pipettors and using the rAAV-ΔCap-in-cis acceptor for cloning thelibraries was sufficient to prevent contamination from AAV9 or AAV9RX/A.

The AAV2/9 REP-AAP helper plasmid was constructed by introducing 5 stopcodons into the coding sequence of the VP reading frame of the AAV9 geneat AAs 6, 10, 142, 148, 216 (VP1 numbering). The stop codon at AA216 wasdesigned such that it did not disrupt the coding sequence of the AAPprotein, which is encoded within an alternative reading frame.

Library Generation

The 452-8r and 588i library fragments were generated by PCR using Q5 HotStart, High-Fidelity DNA Polymerase (NEB). A schematic showing theapproximate primer binding sites and the primer sequences are given inFIGS. 27A and 27C, respectively. To facilitate cloning of the PCRfragments comprising the capsid library sequences into a rAAV genome,the rAAV-cap-in-cis plasmid was modified to introduce two uniquerestriction sites, XbaI and AgeI, within the capsid sequence flankingthe region that was replaced by the PCR fragment. The insertion of theXbaI site caused a K449R mutation and the mutations required to insertthe AgeI site were silent. To prevent contamination of the libraries by“wild type” AAV9R X/A capsid, the coding region between the XbaI andAgeI sites was eliminated from the rAAV-cap-in-cis plasmid to createrAAV-Cap-in-cis.

To generate the rAAV based library, the PCR products containing thelibrary and the XbaI and AgeI digested cap-in-cis acceptor vector wereassembled using Gibson Assembly. The reaction products were then treatedwith PS DNase (Epicentre) to eliminate any unassembled fragments. Thisreaction typically yielded over 100 ng of assembled plasmid (as definedby the amount of DNA remaining after a PS DNase digestion step). 100 ngis sufficient to transfect 10 150 mm dishes at 10 ng/dish.

Virus Production and Purification

Recombinant AAVs were generated by triple transfection of 293 T cellsusing PEI. Viral particles were harvested from the media at 72 h posttransfection and from the cells and media at 120 h. Virus present in themedia was concentrated by precipitation with 8% poly(ethylene glycol)and 500 mM sodium chloride (Ayuso et al 2010) and then the precipitatedvirus was added to the lysates prepared from the collected cells. Theviruses were purified over iodixanol (Optiprep, Sigma) step gradients(15%, 25%, 40% and 60% as described by Zolotukhin et al 1999). Viruseswere concentrated and formulated in PBS. Virus titers were determined bymeasuring the number of DNaseI-resistant vector genome copies (VGs)using qPCR and the linearized genome plasmid as a control (Gray et al2011).

For capsid library virus generation, two modifications were made to thevirus production protocol to reduce the production of mosaic capsidsthat could arise from the presence of multiple capsid sequences in thesame cell. First, only 10 ng per dish of AAV-Cap9-in-cis library vectorper dish was transfected to insure that the vast majority of transfectedcells only received one capsid variant sequence. Second, the virus wascollected earlier (48 h and 60 hours, instead of 72 h and 120 h asabove) to minimize the secondary transduction of the producer cells withthe rAAV library virus released into the medium.

In Vivo Selection

For the selections in GFAP-Cre mice, 1×10¹¹ vg of the capsid librarieswere injected IV (retro-orbital route) into adult Cre+ mice. Seven oreight days post-injection, mice were euthanized and the brain and spinalcord were collected. Vector DNA was recovered from one hemisphere of thebrain and half of the spinal cord using 4-5 ml of Trizol (Invitrogen).For the selections in TH-Cre mice, 8×10⁹ vg of each capsid was injectedby intracranially using the stereotaxic coordinates 0.7 mm rostral, 2.0mm lateral and 3.0 mm ventral from bregma. 10 days later, the regioncontaining the substantia nigra was collected and the tissue washomogenized in 1 ml of Trizol. For virus DNA isolation, themanufacture's RNA extraction protocol was followed (the upper aqueous,RNA-containing fraction collected). In addition to RNA, it was foundthat this fraction also contains a significant portion of the viralgenome as well as some mitochondrial DNA. RNA was eliminated by treatingthe samples with 1 ul of RNase (Qiagen) at 37 C overnight. The Crerecombination-dependent PCR strategy involved a two-step amplificationstrategy (FIG. 27A-27E). Sequence recovery was first performed in aCre-dependent manner using the primers 9CAPF and CDF (FIG. 27A-27E). PCRwas performed for 20-26 cycles of 95 C for 20 sec, 60 C for 20 sec and72 C for 30 sec using Q5 Hot Start High-fidelity DNA Polymerase. The PCRproduct was then diluted 1:10-1:100 and then used as a template for asecond, PCR reaction using primer XF and AR that generated a shorterfragment that was cloned back into the rAAV-delta-cap-in-cis acceptorconstruct as described above. 1 ul of the Gibson Assembly reactions wasthen diluted 1:10 and transformed into Sure2 competent cells (Agilent)as directed by the manufacturer to generate individual clones forsequencing.

Clones that showed evidence of enrichment were cut with BsiWI and AgeIand ligated into a custom 2/9R-X/A rep/cap helper also cut with BsiWIand AgeI and then transformed into DH5alpha competent cells (NEB). Theresulting rep/cap plasmids carrying the novel variant sequences, orAAV2/9 rep/cap as a control, were then used to package a rAAV genomecontaining a dual eGFP-2A-luciferase reporter cassette driven by aubiquitous CAG promoter (rAAV-CAG-eGFP-2A-Luc-WPRE-SV40 pA) for the IVinjected variants (AAV-PHP.A and AAV-PHP.B) or a similar vector lackingthe Luc gene (rAAV-CAG-eGFP-WPRE-SV40 pA) for the intracranialinjections (AAV-PHP.R2).

Tissue Preparation and Immunostaining

Mice were anesthetized with Nembutal and transcardially perfused firstwith 0.1 M phosphate buffer (PB), pH 7.4 and then with freshly prepared4% paraformaldehyde in PB. Brains were postfixed overnight and thensectioned by vibratome or cryoprotected and sectioned by cryostat.Immunostaining was performed on the floating sections by dilutingprimary and secondary antibodies in PBS containing 10% goat or donkeyserum 0.5% Triton X-100 or no detergent (GAD67 staining). Primaryantibodies used were rabbit anti-GFP (1:1000; Invitrogen), chickenanti-GFP (1:1000; Abcam), mouse anti-CC1 (1:200; Calbiochem), rabbitanti-GFAP (1:1000; Dako), mouse anti-NeuN (1:500; Millipore), rabbitanti IbaI (1:500; Biocare Medical), mouse anti-Calbindin (1:200; Sigma),rabbit anti-Calretinin (1:1000; Chemicon), mouse anti-GAD67 (1:1000;Millipore), mouse anti-Parvalbumin (1:1000). Primary antibodiesincubations were performed for 16-24 hours at room temperature. Thesections washed and incubated with secondary antibodies conjugated toAlexa 568 (1:1000; Invitrogen) for 2-16 hours.

Tissue Clearing

Mice were perfused via peristaltic pump through the left ventricle withphosphate buffer (PB) followed by an initial perfusion with 60-80 mL of4% PFA in PB at a flow rate of 14 mL per minute. The flow rate was thenreduced to 2-3 mL per minute and continued for 2 hours at roomtemperature. The mice were then placed in individual custom-builtperfusion chambers and perfused with 200 mL of recycling 4% acrylamidein PB at the same flow rate at room temperature overnight followed by a2-hour perfusion flush with PB to remove residual polymers/monomers inthe vasculature. The polymerization process was initiated placing thechambers in a 42° C. water bath and delivering, by perfusion at the sameflow rate, 200 mL of recycling, degassed 0.25% VA-044 initiator in PB.After polymerization was complete, the mice were perfused with aclearing solution of 8% SDS in 0.1M PB, pH 7.5 for 7 days. The SDScontaining solution was changed two times and then flushed by theperfusion of roughly 2 L of nonrecirculating PB overnight. Tissuesamples cleared of lipids were incubated in RIMS solution (Yang et al.2014) until imaging (at least one week for optimal transparency ofunsectioned mouse brain tissue). The samples were then mounted in RIMSand enclosed with a coverglass on a slide using an appropriate thicknessspacer (iSpacer, SunJin Lab Co.). Images were taken with a Zeiss LSM 780single-photon microscope. 3 dimensional image reconstructions wereperformed using Imaris imaging software (Bitplane).

Vector Biodistribution

Mice were injected IV with 1×10¹¹ VG of arAAV-CAG-GFP2A-Luc-WPRE-SV40-pA vector packaged into the indicatedcapsids. 25 days later, the mice were euthanized and tissues andindicated brain regions were collected and frozen at −80 C. DNA wasisolated using Qiagen DNeasy Blood and Tissue kit. Vector genomes weredetected using PCR primers that bind to the WPRE element and werenormalized to mouse genomes using primers specific to the mouse glucagongene. Absolute quantification was performed by comparing unknown samplesto serial dilutions of standards of known concentration.

Example 9 Method of Treatment Employing Targeting Proteins

A subject having a disorder that can be treated by the application of anucleic acid to be expressed within a subject is identified. The subjectis then administered a first amount of a vector that includes thepolynucleotide to be expressed. The polynucleotide encodes for atherapeutic protein. The vector will include a capsid protein thatincludes a targeting protein section that is SEQ ID NO: 1, so as toallow proper targeting of the protein to be expressed to the appropriatesystem within the subject. If needed, the subject is administered asecond or third dose of the vector, until a therapeutically effectiveamount of the protein to be expressed is expressed within the subject inthe appropriate system.

Example 10 Method of Treatment of Huntington's Disease

A subject having Huntington's disease is identified. The subject is thenadministered a first amount of a vector that includes the polynucleotideto be expressed. The polynucleotide encodes for a therapeutic protein.The vector will include a capsid protein that includes a targetingprotein section that is SEQ ID NO: 1, so as to allow proper targeting ofthe protein to be expressed to the nervous system within the subject. Ifneeded, the subject is administered a second or third dose of thevector, until a therapeutically effective amount of the protein to beexpressed is expressed within the subject in the nervous system.

Example 11 Method of Treatment of Huntington's Disease

A subject having Huntington's disease is identified. The subject is thenadministered a first amount of a vector that includes a polynucleotidethat encodes for a small non-coding RNA (small hairpin RNA (shRNA) ormicroRNA (miRNA)) configured to reduce expression of the Huntingtinprotein by its sequence). The vector will include a capsid protein thatincludes a targeting protein of SEQ ID NO: 1, so as to allow propertargeting of the said polynucleotide to the nervous system. If needed,the subject is administered a second or third dose of the vector, untila therapeutically effective amount of the small non-coding RNA isexpressed the subject in the nervous system.

Example 12 Method of Treatment

A subject having Huntington's disease is identified. The subject is thensystemically administered a first amount of a vector that includes apolynucleotide that encodes for a Zinc finger protein (ZFP) engineeredto represses the transcription of the Huntingtin (HTT) gene. The vectorwill include a capsid protein that includes a targeting protein of SEQID NO: 1 or any of the targeting proteins in FIG. 31, so as to allowproper targeting of the ZFP to the nervous system, among other organs.If needed, the subject is administered a second or third dose of thevector, until a therapeutically effective amount of the ZFP is expressedthe subject in the nervous system.

Example 13 Method of Treatment

A subject having Huntington's disease is identified. The subject is thensystemically administered a first amount of a vector that includes apolynucleotide that encodes for a small non-coding RNA (small hairpinRNA (shRNA) or microRNA (miRNA)) designed by one skilled in the art toreduce expression of the Huntingtin protein. The vector will include acapsid protein that includes a targeting protein of SEQ ID NO: 1 or anyof the targeting proteins in FIG. 31, so as to allow proper targeting ofthe polynucleotide to the nervous system, among other organs. If needed,the subject is administered a second or third dose of the vector, untila therapeutically effective amount of the small non-coding RNA isexpressed the subject in the nervous system.

Example 14 Method of Treatment

A subject having Alzheimer's disease is identified. The subject is thenadministered a first amount of a vector that includes a polynucleotidethat encodes for an anti-Abeta antibodies or antibody fragments. Thevector will include a capsid protein that includes a targeting proteinof SEQ ID NO: 1 or any of the targeting proteins in FIG. 31, so as toallow proper targeting of the antibody or antibody fragment to beexpressed to the nervous system. If needed, the subject is administereda second or third dose of the vector, until a therapeutically effectiveamount of the antibody or antibody fragment is expressed the subject inthe nervous system.

Example 15 Method of Treatment

A subject having Alzheimer's disease is identified. The subject is thenadministered a first amount of a vector that includes a polynucleotidethat encodes for an apolipoprotein E (ApoE) protein, preferably thehuman apoE polypeptide apoE2 or modified variant of apoE2. The vectorwill include a capsid protein that includes a targeting protein of SEQID NO: 1 or any of the targeting proteins in FIG. 31, so as to allowproper targeting of the antibody or antibody fragment to be expressed tothe nervous system. If needed, the subject is administered a second orthird dose of the vector, until a therapeutically effective amount ofthe ApoE protein is expressed the subject in the nervous system.

Example 16 Method of Treatment

A subject having spinal muscular atrophy (SMA) is identified. Thesubject is then administered a first amount of a vector that includes apolynucleotide that encodes for a survival motor neuron 1 (SMN1)polypeptide. The vector will include a capsid protein that includes atargeting protein of SEQ ID NO: 1 or any of the targeting proteins inFIG. 31, so as to allow proper targeting of the SMN protein to beexpressed to the nervous system. If needed, the subject is administereda second or third dose of the vector, until a therapeutically effectiveamount of the SMN protein is expressed the subject in the nervoussystem.

Example 17 Method of Treatment

A subject having Friedreich's ataxia is identified. The subject is thensystemically administered a first amount of a vector that includes apolynucleotide that encodes for a frataxin protein. The vector willinclude a capsid protein that includes a targeting protein of SEQ ID NO:1 or any of the targeting proteins in FIG. 31, so as to allow propertargeting of the frataxin protein to be expressed to the nervous systemand heart, among other organs. If needed, the subject is administered asecond or third dose of the vector, until a therapeutically effectiveamount of the frataxin protein is expressed the subject in the nervoussystem and heart.

Example 18 Method of Treatment

A subject having Pompe disease is identified. The subject is thensystemically administered a first amount of a vector that includes apolynucleotide that encodes for an acid alpha-glucosidase (GAA) protein.The vector will include a capsid protein that includes a targetingprotein of SEQ ID NO: 1 or any of the targeting proteins in FIG. 31, soas to allow proper targeting of the GAA protein to be expressed to thenervous system and heart, among other organs. If needed, the subject isadministered a second or third dose of the vector, until atherapeutically effective amount of the GAA protein is expressed thesubject in the nervous system and heart.

Example 19 Method of Treatment

A subject having Late Infantile neuronal ceroid lipofuscinosis (LINCL)is identified. The subject is then systemically administered a firstamount of a vector that includes a CLN2 polynucleotide that encodes forthe tripeptidyl peptidase 1 protein. The vector will include a capsidprotein that includes a targeting protein of SEQ ID NO: 1 or any of thetargeting proteins in FIG. 31, so as to allow proper targeting of thetripeptidyl peptidase 1 protein to be expressed to the nervous system.If needed, the subject is administered a second or third dose of thevector, until a therapeutically effective amount of the tripeptidylpeptidase 1 protein is expressed the subject in the nervous system.

Example 20 Method of Treatment

A subject having the Juvenile NCL form of Batten disease is identified.The subject is then systemically administered a first amount of a vectorthat includes a CLN3 polynucleotide that encodes for the batteninprotein. The vector will include a capsid protein that includes atargeting protein of SEQ ID NO: 1 or any of the targeting proteins inFIG. 31, so as to allow proper targeting of the battenin protein to beexpressed to the nervous system. If needed, the subject is administereda second or third dose of the vector, until a therapeutically effectiveamount of the battenin protein is expressed the subject in the nervoussystem.

Example 21 Method of Treatment

A subject having Canavan disease is identified. The subject is thensystemically administered a first amount of a vector that includes anASPA polynucleotide that encodes for the aspartoacylase protein. Thevector will include a capsid protein that includes a targeting proteinof SEQ ID NO: 1 or any of the targeting proteins in FIG. 31, so as toallow proper targeting of the aspartoacylase protein to be expressed tothe nervous system. If needed, the subject is administered a second orthird dose of the vector, until a therapeutically effective amount ofthe aspartoacylase protein is expressed the subject in the nervoussystem.

Example 22 Method of Treatment

A subject having Parkinson's disease is identified. The subject is thensystemically administered a first amount of one or more vectors thateach includes one or more polynucleotide(s) that encode an enzyme(s)necessary for the increased production of dopamine from non-dopaminergiccells. The vector will include a capsid protein that includes atargeting protein of SEQ ID NO: 1 or any of the targeting proteins inFIG. 31, so as to allow proper targeting of said enzyme(s) to beexpressed to the nervous system. If needed, the subject is administereda second or third dose of the vector, until a therapeutically effectiveamount of the enzyme(s) is expressed the subject in the nervous system.

Example 23 Method of Treatment

A subject having Parkinson's disease is identified. The subject is thensystemically administered a first amount of a vector that includes apolynucleotide that encode a modified, aggregation-resistant form ofalpha-synuclein protein that reduces the aggregation of endogenousalpha-synuclein. The vector will include a capsid protein that includesa targeting protein of SEQ ID NO: 1 or any of the targeting proteins inFIG. 31, so as to allow proper targeting of the aggregation-resistantalpha-synuclein protein to be expressed to the nervous system. Ifneeded, the subject is administered a second or third dose of thevector, until a therapeutically effective amount of the protein isexpressed the subject in the nervous system.

Example 24 Method of Treatment

A subject having amyotrophic lateral sclerosis or frontal dementiacaused by a mutation in C9ORF72 is identified. The subject is thenadministered a first amount of a vector that includes a polynucleotidethat encodes a non-coding RNA(s) that reduce nuclear RNA foci caused bythe hexanucleotide expansion (GGGGCC) in the subjects cells. The vectorwill include a capsid protein that includes a targeting protein of SEQID NO: 1 or any of the targeting proteins in FIG. 31, so as to allowproper targeting of the RNA(s) to be expressed to the nervous system. Ifneeded, the subject is administered a second or third dose of thevector, until a therapeutically effective amount of the RNA(s) isexpressed the subject in the nervous system.

Example 25 Method of Treatment

A subject having multiple sclerosis is identified. The subject is thensystemically administered a first amount of a vector that includes apolynucleotide that encode a trophic or immunomodulatory factor, forexample leukemia inhibitory factor (LIF) or ciliary eurotrophic factor(CNTF). The vector will include a capsid protein that includes atargeting protein of SEQ ID NO: 1 or any of the targeting proteins inFIG. 31, so as to allow proper targeting of the said factor to beexpressed to the nervous system. If needed, the subject is administereda second or third dose of the vector, until a therapeutically effectiveamount of the factor is expressed the subject in the nervous system.

Example 26 Method of Treatment

A subject having amyotrophic lateral sclerosis caused by SOD1 mutationis identified. The subject is then administered a first amount of avector that includes a polynucleotide that encodes for a smallnon-coding RNA (small hairpin RNA (shRNA) or microRNA (miRNA)) designedby one skilled in the art to reduce expression of mutant SOD1 protein.The vector will include a capsid protein that includes a targetingprotein of SEQ ID NO: 1 or any of the targeting proteins in FIG. 31, soas to allow proper targeting of the small non-coding RNA to be expressedto the nervous system. If needed, the subject is administered a secondor third dose of the vector, until a therapeutically effective amount ofthe small non-coding RNA is expressed the subject in the nervous system.

Additional Embodiments

In some embodiments, provided herein is a CREATE—Cre Recombinase-basedAAV Targeted Evolution platform.

In some embodiments, provided herein is an AAV-PHP.B, which allows Broadgene delivery to CNS neurons and glia via the vasculature.

In some embodiments, provided herein is an AAV-PHP.R2, which allowsRapid Retrograde transduction in the CNS.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety. To the extent that any of the definitionsor terms provided in the references incorporated by reference differfrom the terms and discussion provided herein, the present terms anddefinitions control.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and examples detail certain preferred embodiments of theinvention and describe the best mode contemplated by the inventors. Itwill be appreciated, however, that no matter how detailed the foregoingmay appear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

1. An AAV vector comprising an amino acid sequence that comprises atleast 4 contiguous amino acids from a sequence selected from the groupconsisting of ILGTGTS (SEQ ID NO: 55), TRTNPEA (SEQ ID NO: 56), ILGTGTS(SEQ ID NO: 57); NGGTSSS (SEQ ID NO: 58), YTLSQGW (SEQ ID NO: 60),SVSKPFL (SEQ ID NO: 28), and FTLTTPK (SEQ ID NO: 29).
 2. The AAV vectorof claim 1, wherein the amino acid sequence is part of a capsid proteinof the AAV vector.
 3. The AAV vector of claim 1, wherein the amino acidsequence is inserted between AA588-589 of an AAV9 capsid proteincomprising SEQ ID NO:
 2. 4. The AAV vector of claim 1, wherein the aminoacid sequence is inserted between AA586-592 of an AAV9 capsid proteincomprising SEQ ID NO:
 2. 5. (canceled)
 6. The AAV vector of claim 1,wherein the amino acid sequence consists of an amino acid sequenceselected from the group consisting of LGTGTS (SEQ ID NO: 55), TRTNPEA(SEQ ID NO: 56), ILGTGTS (SEQ ID NO: 57); NGGTSSS (SEQ ID NO: 58),YTLSQGW (SEQ ID NO: 60), SVSKPFL (SEQ ID NO: 28), and FTLTTPK (SEQ IDNO: 29).
 7. The AAV vector of claim 1, wherein the vector comprises anrAAV genome.
 8. The AAV vector of claim 7, wherein the rAAV genomecomprises at least one inverted terminal repeat.
 9. The AAV vector ofclaim 7, wherein the rAAV genome comprises a rep gene.
 10. The AAVvector of claim 7, wherein the rAAV genome comprises a nucleic acidencoding VP3.
 11. An AAV capsid protein comprising an amino acidsequence that comprises at least four contiguous amino acid sequencesfrom a sequence selected from the group consisting of: LGTGTS (SEQ IDNO: 55), TRTNPEA (SEQ ID NO: 56), ILGTGTS (SEQ ID NO: 57); NGGTSSS (SEQID NO: 58), YTLSQGW (SEQ ID NO: 60), SVSKPFL (SEQ ID NO: 28), andFTLTTPK (SEQ ID NO: 29).
 12. The AAV capsid protein of claim 11,comprising the amino acid sequence of SEQ ID NO: 55, SEQ ID NO: 58, SEQID NO: 60, SEQ ID NO: 28 or SEQ ID NO:
 29. 13. The AAV capsid protein ofclaim 11, further conjugated to a nanoparticle or second molecule. 14.The AAV capsid protein of claim 11, wherein the AAV capsid protein ispart of an AAV.
 15. The AAV capsid protein of claim 11, wherein the AAVis selected from AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, human isolate hu.31, human isolate hu.32, rhesus isolaterh.8, and rhesus isolate rh.10.
 16. The AAV capsid protein of claim 15,wherein the AAV is an AAV9.
 17. A peptide comprising an amino acidsequence selected from the group consisting of: LGTGTS (SEQ ID NO: 55),TRTNPEA (SEQ ID NO: 56), ILGTGTS (SEQ ID NO: 57); NGGTSSS (SEQ ID NO:58), YTLSQGW (SEQ ID NO: 60), SVSKPFL (SEQ ID NO: 28), and FTLTTPK (SEQID NO: 29).
 18. The peptide of claim 17, consisting essentially of anamino acid sequence selected from the group consisting of: LGTGTS (SEQID NO: 55), TRTNPEA (SEQ ID NO: 56), ILGTGTS (SEQ ID NO: 57); NGGTSSS(SEQ ID NO: 58), YTLSQGW (SEQ ID NO: 60), SVSKPFL (SEQ ID NO: 28), andFTLTTPK (SEQ ID NO: 29).
 19. The peptide of claim 17, linked to ananoparticle.
 20. The peptide of claim 18, covalently linked to aprotein.
 21. The AAV vector of claim 1, wherein the amino acid sequenceis part of an AAV9 capsid protein.
 22. The AAV vector of claim 21,wherein the amino acid sequence is inserted into the unstructuredsurface loop of the AAV9 capsid protein.
 23. The AAV vector of claim 21,wherein the amino acid sequence is inserted into amino acids 586-592,452-458, 262-269, 464-473, 491-495, 546-557, or 659-668 of the AAV9capsid protein comprising the sequence of SEQ ID NO:
 2. 24. The AAVvector of claim 1, wherein the amino acid sequence comprises at least 5contiguous amino acids from SVSKPFL (SEQ ID NO: 28) or FTLTTPK (SEQ IDNO: 29).
 25. The AAV vector of claim 1, wherein the amino acid sequencecomprises at least 6 contiguous amino acids from SVSKPFL (SEQ ID NO: 28)or FTLTTPK (SEQ ID NO: 29).
 26. The AAV vector of claim 1, wherein theamino acid sequence comprises the sequence of SVSKPFL (SEQ ID NO: 28) orFTLTTPK (SEQ ID NO: 29).
 27. The AAV capsid protein of claim 11, whereinthe amino acid sequence comprises at least 5 contiguous amino acid fromSVSKPFL (SEQ ID NO: 28) or FTLTTPK (SEQ ID NO: 29).
 28. The AAV capsidprotein of claim 11, wherein the amino acid sequence comprises at least6 contiguous amino acids from SVSKPFL (SEQ ID NO: 28) or FTLTTPK (SEQ IDNO: 29).
 29. The AAV capsid protein of claim 11, wherein the amino acidsequence comprises the sequence of SVSKPFL (SEQ ID NO: 28) or FTLTTPK(SEQ ID NO: 29).